Bile acid derivatives as FXR/TGR5 agonists and methods of use thereof

ABSTRACT

The present invention provides compounds of Formula I,pharmaceutical compositions comprising these compounds and methods of using these compounds to prevent or treat FXR-mediated or TGR5-mediated diseases or conditions.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/878,329, filed May 19, 2020, which is a continuation of U.S.application Ser. No. 16/222,380, filed Dec. 17, 2018, now U.S. Pat. No.10,696,713, issued Jun. 30, 2020, which is a continuation of U.S.application Ser. No. 14/951,989, filed Nov. 25, 2015, now U.S. Pat. No.10,208,081, issued Feb. 19, 2019, which claims the benefit of U.S.Provisional Application No. 62/084,769, filed on Nov. 26, 2014, and U.S.Provisional Application No. 62/103,374, filed on Jan. 14, 2015. Theentire teachings of the above applications are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates generally to compounds and pharmaceuticalcompositions useful as FXR/TGR5 modulators. Specifically, the presentinvention relates to bile acid derivatives and methods for theirpreparation and use.

BACKGROUND OF THE INVENTION

Farnesoid X Receptor (FXR) is an orphan nuclear receptor initiallyidentified from a rat liver cDNA library (BM. Forman, et al., Cell,1995, 81(5), 687-693) that is most closely related to the insectecdysone receptor. FXR is a member of the nuclear receptor family ofligand-activated transcription factors that includes receptors for thesteroid, retinoid, and thyroid hormones (D J. Mangelsdorf, et al., Cell,1995, 83(6), 841-850). The relevant physiological ligands of FXR arebile acids (D. Parks et al., Science, 1999, 284(5418), 1362-1365). Themost potent one is chenodeoxycholic acid (CDCA), which regulates theexpression of several genes that participate in bile acid homeostasis.Farnesol and derivatives, together called farnesoids, are originallydescribed to activate the rat orthologue at high concentration but theydo not activate the human or mouse receptor. FXR is expressed in theliver, throughout the entire gastrointestinal tract including theesophagus, stomach, duodenum, small intestine, colon, ovary, adrenalgland and kidney. Beyond controlling intracellular gene expression, FXRseems to be also involved in paracrine and endocrine signaling byupregulating the expression of the cytokine Fibroblast Growth Factor (J.Holt et al., Genes Dev., 2003, 17(13), 1581-1591; T. Inagaki et al.,Cell Metab., 2005, 2(4), 217-225).

Small molecule compounds which act as FXR modulators have been disclosedin the following publications: WO 2000/037077, WO 2003/015771, WO2004/048349, WO 2007/076260, WO 2007/092751, WO 2007/140174, WO2007/140183, WO 2008/051942, WO 2008/157270, WO 2009/005998, WO2009/012125, WO 2008/025539, WO 2008/025540, WO 2011/020615, and WO2013/007387.

Further small molecule FXR modulators have been recently reviewed (R. C.Buijsman et al. Curr. Med. Chem. 2005, 12, 1017-1075).

TGR5 receptor is a G-protein-coupled receptor that has been identifiedas a cell-surface receptor that is responsive to bile acids (BAs). Theprimary structure of TGR5 and its responsiveness to bile acids has beenfound to be highly conserved in TGR5 among human, bovine, rabbit, rat,and mouse, and thus suggests that TGR5 has important physiologicalfunctions. TGR5 has been found to be widely distributed in not onlylymphoid tissues but also in other tissues. High levels of TGR5 mRNAhave been detected in placenta, spleen, and monocytes/macrophages. Bileacids have been shown to induce internalization of the TGR5 fusionprotein from the cell membrane to the cytoplasm (Kawamata et al., J.Bio. Chem., 2003, 278, 9435). TGR5 has been found to be identical tohGPCR19 reported by Takeda et al., FEBS Lett. 2002, 520, 97-101.

TGR5 is associated with the intracellular accumulation of cAMP, which iswidely expressed in diverse cell types. While the activation of thismembrane receptor in macrophages decreases pro-inflammatory cytokineproduction, (Kawamata, Y., et al., J. Biol. Chem. 2003, 278, 9435-9440)the stimulation of TGR5 by BAs in adipocytes and myocytes enhancesenergy expenditure (Watanabe, M., et al. Nature. 2006, 439, 484-489).This latter effect involves the cAMP-dependent induction of type 2iodothyronine deiodinase (D2), which by, locally converting T4 into T3,gives rise to increased thyroid hormone activity. Consistent with therole of TGR5 in the control of energy metabolism, female TGR5 knock-outmice show a significant fat accumulation with body weight gain whenchallenged with a high fat diet, indicating that the lack of TGR5decreases energy expenditure and elicits obesity (Maruyama, T., et al.,J. Endocrinol. 2006, 191, 197-205). In addition, and in line with theinvolvement of TGR5 in energy homeostasis, bile acid activation of themembrane receptor has also been reported to promote the production ofglucagon-like peptide 1 (GLP-1) in murine enteroendocrine cell lines(Katsuma, S., Biochem. Biophys. Res. Commun., 2005, 329, 386-390). Onthe basis of all the above observations, TGR5 is an attractive targetfor the treatment of disease e.g., obesity, diabetes and metabolicsyndrome.

In addition to the use of TGR5 agonists for the treatment and preventionof metabolic diseases, compounds that modulate TGR5 modulators are alsouseful for the treatment of other diseases e.g., central nervousdiseases as well as inflammatory diseases (WO 01/77325 and WO 02/84286).Modulators of TGR5 also provide methods of regulating bile acid andcholesterol homeostasis, fatty acid absorption, and protein andcarbohydrate digestion.

There is a need for the development of FXR and/or TGR5 modulators forthe treatment and prevention of disease. The present invention hasidentified compounds, which contain an amino, urea, sulfonyurea orsulfonamide moieties, which modulate FXR and/or TGR as well as methodsof using these compounds to treat disease.

SUMMARY OF THE INVENTION

In one aspect, the invention provides compounds represented by FormulaI, or pharmaceutically acceptable salts, stereoisomers, solvates,hydrates or combinations thereof:

wherein:

-   R_(a) is hydrogen or substituted or unsubstituted —C₁-C₈ alkyl;    preferably R_(a) is hydrogen or methyl; more preferably, R_(a) is    hydrogen;-   R_(b) is selected from the group consisting of:

1) Hydrogen;

2) —C(O)NR₁₀R₁₁;

3) —C(O)NHSO₂R₁; and

4) —SO₂R₁;

-   R₁ is selected from the group consisting of:

1) Halogen;

2) Hydroxyl;

3) Substituted or unsubstituted —C₁-C₈ alkyl;

4) Substituted or unsubstituted —C₂-C₈ alkenyl;

5) Substituted or unsubstituted —C₂-C₈ alkynyl;

6) Substituted or unsubstituted —C₃-C₈ cycloalkyl;

7) Substituted or unsubstituted aryl;

8) Substituted or unsubstituted arylalkyl;

9) Substituted or unsubstituted heterocycloalkyl;

10) Substituted or unsubstituted heteroaryl;

11) Substituted or unsubstituted heteroarylalkyl; and

12) —NR₁₀R₁₁;

-   R₂ is selected from the group consisting of:

1) Hydrogen;

2) Substituted or unsubstituted —C₁-C₈ alkyl;

3) Substituted or unsubstituted —C₂-C₈ alkenyl;

4) Substituted or unsubstituted —C₂-C₈ alkynyl;

5) Substituted or unsubstituted arylalkyl; and

6) Substituted or unsubstituted aryl;

preferably R₂ is hydrogen or methyl.

-   m is selected from 0, 1, 2 and 3, preferably m is 0, 1 or 2.-   R₃ is hydrogen, hydroxyl, —OSO₃H, —OSO₃ ⁻, —OAc, —OPO₃H₂ or —OPO₃    ²⁻, preferably R₃ is hydrogen or hydroxyl.-   R₄ is hydrogen, halogen, CN, N₃, hydroxyl, —OSO₃H, —OSO₃ ⁻, —OAc,    —OPO₃H₂, —OPO₃ ²⁻, —SR₂ or —NHR₂, wherein, R₂ is as defined    previously; preferably R₄ is hydrogen.-   Or R₃ and R₄ are taken together with the carbon atoms to which they    are attached to form —CH═CH— or a cycloalkyl ring or    heterocycloalkyl ring such as, but not limited to cyclopropyl, or    epoxide.-   R₅ and R₆ are independently selected from hydrogen or hydroxyl    protecting group such as, but not limited to acetyl, trimethyl    silyl, or benzyl; preferably R₅ and R₆ are hydrogen.-   R₇ is selected from the group consisting of:    -   1) Hydrogen;    -   2) Halogen;    -   3) Substituted or unsubstituted —C₁-C₈ alkyl;    -   4) Substituted or unsubstituted —C₂-C₈ alkenyl;    -   5) Substituted or unsubstituted —C₂-C₈ alkynyl; and    -   6) Substituted or unsubstituted —C₃-C₈ cycloalkyl;    -   preferably R₇ is C₁-C₄-alkyl, more preferably R₇ is ethyl;-   R₁₀ and R₁₁ are each independently selected from hydrogen,    substituted or unsubstituted —C₁-C₈ alkyl, substituted or    unsubstituted —C₂-C₈ alkenyl, substituted or unsubstituted —C₂-C₈    alkynyl, substituted or unsubstituted —C₃-C₈ cycloalkyl, substituted    or unsubstituted heterocycloalkyl, or R₁₀ and R₁₁ are taken together    with the nitrogen atom they attached to form a heterocyclic ring;    preferably, R₁₁ is hydrogen.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundor combination of compounds of the present invention, or apharmaceutically acceptable salt form, stereoisomer, solvate, hydrate orcombination thereof, in combination with a pharmaceutically acceptablecarrier or excipient.

In another embodiment, the present invention provides a method for theprevention or treatment of an FXR mediated disease or condition. Themethod comprises administering a therapeutically effective amount of acompound of the invention. The present invention also provides the useof a compound of the invention for the preparation of a medicament forthe prevention or treatment of an FXR mediated disease or condition.

In yet another embodiment, the present invention provides a method forthe prevention or treatment of a TGR5 mediated disease or condition. Themethod comprises administering a therapeutically effective amount of acompound of the invention. The present invention also provides the useof a compound of the invention for the preparation of a medicament forthe prevention or treatment of a TGR5 mediated disease or condition.

In certain embodiments, a disease that involves modulation of the TGR5receptor is selected from metabolic disease, inflammatory disease, liverdisease, autoimmune disease, cardiac disease, kidney disease, cancer,and gastrointestinal disease.

DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention is a compound represented by FormulaI as described above, or a pharmaceutically acceptable salt, hydrate,solvate, ester or prodrug thereof. In preferred compounds of Formula I,R₂, R₃, R₄, R₅, and R₆ are each hydrogen and R₇ is ethyl.

In one embodiment, the invention provides compounds of Formula I′,

or a pharmaceutically acceptable salt or prodrug thereof, wherein:R_(a) is hydrogen;R_(b) is —C(O)NHSO₂R₁;R₁ is selected from the group consisting of:

-   -   1) Halogen;    -   2) Hydroxyl;    -   3) Substituted or unsubstituted —C₁-C₈ alkyl;    -   4) Substituted or unsubstituted —C₂-C₈ alkenyl;    -   5) Substituted or unsubstituted —C₂-C₈ alkynyl;    -   6) Substituted or unsubstituted —C₃-C₈ cycloalkyl;    -   7) Substituted or unsubstituted aryl;    -   8) Substituted or unsubstituted alkylaryl;    -   9) Substituted or unsubstituted heterocycloalkyl;    -   10) Substituted or unsubstituted heteroaryl;    -   11) Substituted or unsubstituted alkylheteroaryl; and    -   12) —NR₁₀R₁₁;        R₂ is selected from the group consisting of:    -   1) Hydrogen;    -   2) Substituted or unsubstituted —C₁-C₈ alkyl;    -   3) Substituted or unsubstituted —C₂-C₈ alkenyl;    -   4) Substituted or unsubstituted —C₂-C₈ alkynyl;    -   5) Substituted or unsubstituted alkylaryl; and    -   6) Substituted or unsubstituted aryl;

Preferably R₂ is hydrogen or methyl;

m is selected from 0, 1, 2 and 3, preferably m is from 0, 1 or 2.

R₃ is hydrogen, hydroxyl, —OSO₃H, —OSO₃ ⁻, —OAc, —OPO₃H₂ or —OPO₃ ²⁻,preferably R₃ is hydrogen or hydroxyl.

R₄ is hydrogen, halogen, CN, N₃, hydroxyl, —OSO₃H, —OSO₃ ⁻, —OAc,—OPO₃H₂, —OPO₃ ²⁻, —SR₂ or —NHR₂, wherein, R₂ is as defined previously;preferably R₄ is hydrogen. Or R₃ and R₄ are taken together with thecarbon atoms they attached form —CH═CH— or a cycloalkyl ring orheterocycloalkyl ring such as, but not limited to cyclopropyl, orepoxide.R₅ and R₆ are independently selected from hydrogen or hydroxylprotecting group such as, but not limited to acetyl, trimethylsilyl, orbenzyl; preferably R₅ and R₆ are hydrogen.R₇ is selected from the group consisting of:

1) Hydrogen;

2) Halogen;

3) Substituted or unsubstituted —C₁-C₈ alkyl;

4) Substituted or unsubstituted —C₂-C₈ alkenyl;

5) Substituted or unsubstituted —C₂-C₈ alkynyl; and

6) Substituted or unsubstituted —C₃-C₈ cycloalkyl;

preferably R₇ is C₁-C₄-alkyl, more preferably R₇ is ethyl;

R₁₀ and R₁₁ are each independently selected from hydrogen, substitutedor unsubstituted —C₁-C₈ alkyl, substituted or unsubstituted —C₂-C₈alkenyl, Substituted or unsubstituted —C₂-C₈ alkynyl, substituted orunsubstituted —C₃-C₈ cycloalkyl, or R₁₀ and R₁₁, taken together with thenitrogen atom to which they are attached, form a heterocyclic ring;preferably R₁₁ is hydrogen.

In preferred embodiments, the compounds of the invention have thestereochemistry set forth in Formula IA:

where m, R_(a), R_(b), R₂, R₃, R₄, R₅, R₆, and R₇ have the meaningsgiven for these variables in Formula I or Formula I′.

A second embodiment of the invention is a compound represented byFormula II or a pharmaceutically acceptable salt, hydrate, solvate,ester or prodrug thereof:

wherein R_(a), R_(b), R₂, R₃, R₄, R₇ and m are as previously defined inFormula I or I′.

A third embodiment of the invention is a compound represented by FormulaIII or a pharmaceutically acceptable salt, hydrate, solvate, ester orprodrug thereof:

wherein R_(a), R_(b), R₂, R₃, R₇ and m are as previously defined inFormula I or I′.

Illustrative structures of Formula (III) can be represented, but notlimited, by formula (III-1˜III-54), where R₁, R₇, R₁₀ and m are aspreviously defined in Formula I or I′:

A fourth embodiment of the invention is a compound represented byFormula IV-A, IV-B, IV-C, or IV-D or a pharmaceutically acceptable salt,solvate, hydrate, ester or prodrug thereof,

wherein R₁ and m are as previously defined in Formula I or I′.

In certain embodiments of the compounds of the invention, R₁ isC₁-C₄-alkyl, halogenated C₁-C₄-alkyl, C₁-C₄-alkenyl, phenyl-C₁-C₄-alkyl,substituted or unsubstituted C₃-C₆-cycloalkyl,C₁-C₆-cycloalkyl-C₁-C₄-alkyl, 5- or 6-membered heterocycloalkyl, amino,substituted or unsubstituted phenyl or halogen.

In certain embodiments of the compounds of the invention, R₁ is ethyl,butyl, t-butyl, propyl, benzyl, vinyl, allyl, CF₃,

or fluoro; or R₁ is methyl, isopropyl or phenyl. In certain embodimentsof the compounds of the invention, R₁ is dimethylamino orp-tert-butylphenyl.

In certain embodiments of the invention, R₁ is selected from the groupsset forth in the table below:

In certain embodiments of the compounds of the invention, R₁₁ ishydrogen and R₁₀ is hydrogen, C₁-C₄-alkyl, halogenated C₁-C₄-alkyl,C₁-C₄-alkenyl, phenyl-C₁-C₄-alkyl, substituted or unsubstitutedC₃-C₆-cycloalkyl, C₁-C₆-cycloalkyl-C₁-C₄-alkyl, 5- or 6-memberedheterocycloalkyl, or substituted or unsubstituted phenyl.

In certain embodiments of the compounds of the invention, R₁₁ ishydrogen and R₁₀ is hydrogen, methyl, ethyl, isopropyl, butyl, t-butyl,propyl, benzyl, vinyl, allyl, CF₃,

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 1 to 75 in Table 1) according toFormula IV-A, wherein, R₁ and m are delineated for each compound inTable 1.

TABLE 1 Compound m R₁ 1 0 Methyl 2 0 Ethyl 3 0 Isopropyl 4 0 Butyl 5 0t-Butyl 6 0 Propyl 7 0 Benzyl 8 0 Vinyl 9 0 Allyl 10 0 CF₃ 11 0

12 0

13 0

14 0

15 0

16 0

17 0 NH₂ 18 0

19 0

20 0

21 0

22 0

23 0

24 0

25 0 F 26 1 Methyl 27 1 Ethyl 28 1 Isopropyl 29 1 Butyl 30 1 t-Butyl 311 Propyl 32 1 Benzyl 33 1 Vinyl 34 1 Allyl 35 1 CF₃ 36 1

37 1

38 1

39 1

40 1

41 1

42 1 NH₂ 43 1

44 1

45 1

46 1

47 1

48 1

49 1

50 1 F 51 2 Methyl 52 2 Ethyl 53 2 Isopropyl 54 2 Butyl 55 2 t-Butyl 562 Propyl 57 2 Benzyl 58 2 Vinyl 59 2 Allyl 60 2 CF₃ 61 2

62 2

63 2

64 2

65 2

66 2

67 2 NH₂ 68 2

69 2

70 2

71 2

72 2

73 2

74 2

75 2 F

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 76 to 150 in Table 2) accordingto Formula IV-B, wherein, R₁ and m are delineated for each compound inTable 2.

TABLE 2 Compound m R₁ 76 0 Methyl 77 0 Ethyl 78 0 Isopropyl 79 0 Butyl80 0 t-Butyl 81 0 Propyl 82 0 Benzyl 83 0 Vinyl 84 0 Allyl 85 0 CF₃ 86 0

87 0

88 0

89 0

90 0

91 0

92 0 NH₂ 93 0

94 0

95 0

96 0

97 0

98 0

99 0

100 0 F 101 1 Methyl 102 1 Ethyl 103 1 Isopropyl 104 1 Butyl 105 1t-Butyl 106 1 Propyl 107 1 Benzyl 108 1 Vinyl 109 1 Allyl 110 1 CF₃ 1111

112 1

113 1

114 1

115 1

116 1

117 1 NH₂ 118 1

119 1

120 1

121 1

122 1

123 1

124 1

125 1 F 126 2 Methyl 127 2 Ethyl 128 2 Isopropyl 129 2 Butyl 130 2t-Butyl 131 2 Propyl 132 2 Benzyl 133 2 Vinyl 134 2 Allyl 135 2 CF₃ 1362

137 2

138 2

139 2

140 2

141 2

142 2 NH₂ 143 2

144 2

145 2

146 2

147 2

148 2

149 2

150 2 F

A fifth embodiment of the invention is a compound represented by FormulaV-A and V-B or a pharmaceutically acceptable salt, solvate, hydrate,ester or prodrug thereof.

wherein R₁ and m are as previously defined in Formula I or I′.

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 151 to 225 in Table 3) accordingto Formula V-A, wherein, R₁ and m are delineated for each compound inTable 3.

TABLE 3 Compound m R₁ 151 0 Methyl 152 0 Ethyl 153 0 Isopropyl 154 0Butyl 155 0 t-Butyl 156 0 Propyl 157 0 Benzyl 158 0 Vinyl 159 0 Allyl160 0 CF₃ 161 0

162 0

163 0

164 0

165 0

166 0

167 0 NH₂ 168 0

169 0

170 0

171 0

172 0

173 0

174 0

175 0 F 176 1 Methyl 177 1 Ethyl 178 1 Isopropyl 179 1 Butyl 180 1t-Butyl 181 1 Propyl 182 1 Benzyl 183 1 Vinyl 184 1 Allyl 185 1 CF₃ 1861

187 1

188 1

189 1

190 1

191 1

192 1 NH₂ 193 1

194 1

195 1

196 1

197 1

198 1

199 1

200 1 F 201 2 Methyl 202 2 Ethyl 203 2 Isopropyl 204 2 Butyl 205 2t-Butyl 206 2 Propyl 207 2 Benzyl 208 2 Vinyl 209 2 Allyl 210 2 CF₃ 2112

212 2

213 2

214 2

215 2

216 2

217 2 NH₂ 218 2

219 2

220 2

221 2

222 2

223 2

224 2

225 2 F

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 226 to 300 in Table 4) accordingto Formula V-B, wherein, R₁ and m are delineated for each compound inTable 4.

TABLE 4 Compound m R₁ 226 0 Methyl 227 0 Ethyl 228 0 Isopropyl 229 0Butyl 230 0 t-Butyl 231 0 Propyl 232 0 Benzyl 233 0 Vinyl 234 0 Allyl235 0 CF₃ 236 0

237 0

238 0

239 0

240 0

241 0

242 0 NH₂ 243 0

244 0

245 0

246 0

247 0

248 0

249 0

250 0 F 251 1 Methyl 252 1 Ethyl 253 1 Isopropyl 254 1 Butyl 255 1t-Butyl 256 1 Propyl 257 1 Benzyl 258 1 Vinyl 259 1 Allyl 260 1 CF₃ 2611

262 1

263 1

264 1

265 1

266 1

267 1 NH₂ 268 1

269 1

270 1

271 1

272 1

273 1

274 1

275 1 F 276 2 Methyl 277 2 Ethyl 278 2 Isopropyl 279 2 Butyl 280 2t-Butyl 281 2 Propyl 282 2 Benzyl 283 2 Vinyl 284 2 Allyl 285 2 CF₃ 2862

287 2

288 2

289 2

290 2

291 2

292 2 NH₂ 293 2

294 2

295 2

296 2

297 2

298 2

299 2

300 2 F

A sixth embodiment of the invention is a compound represented by FormulaVI-A or VI-B or a pharmaceutically acceptable salt, solvate, hydrate,ester or prodrug thereof:

wherein R₁₀ and m are as previously defined in Formula I or I′.

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 301 to 375 in Table 5) accordingto Formula VI-A, wherein, R₁₀ and m are delineated for each compound inTable 5.

TABLE 5 Compound m R₁₀ 301 0 Methyl 302 0 Ethyl 303 0 Isopropyl 304 0Butyl 305 0 t-Butyl 306 0 Propyl 307 0 Benzyl 308 0 Vinyl 309 0 Allyl310 0 CF₃ 311 0

312 0

313 0

314 0

315 0

316 0

317 0 H 318 0

319 0

320 0

321 0

322 0

323 0

324 0

325 0

326 1 Methyl 327 1 Ethyl 328 1 Isopropyl 329 1 Butyl 330 1 t-Butyl 331 1Propyl 332 1 Benzyl 333 1 Vinyl 334 1 Allyl 335 1 CF₃ 336 1

337 1

338 1

339 1

340 1

341 1

342 1 H 343 1

344 1

345 1

346 1

347 1

348 1

349 1

350 1

351 2 Methyl 352 2 Ethyl 353 2 Isopropyl 354 2 Butyl 355 2 t-Butyl 356 2Propyl 357 2 Benzyl 358 2 Vinyl 359 2 Allyl 360 2 CF₃ 361 2

362 2

363 2

364 2

365 2

366 2

367 2 H 368 2

369 2

370 2

371 2

372 2

373 2

374 2

375 2

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 376 to 450 in Table 6) accordingto Formula VI-B, wherein, R₁₀ and m are delineated for each compound inTable 6.

TABLE 6 Compound m R₁₀ 376 0 Methyl 377 0 Ethyl 378 0 Isopropyl 379 0Butyl 380 0 t-Butyl 381 0 Propyl 382 0 Benzyl 383 0 Vinyl 384 0 Allyl385 0 CF₃ 386 0

387 0

388 0

389 0

390 0

391 0

392 0 H 393 0

394 0

395 0

396 0

397 0

398 0

399 0

400 0

401 1 Methyl 402 1 Ethyl 403 1 Isopropyl 404 1 Butyl 405 1 t-Butyl 406 1Propyl 407 1 Benzyl 408 1 Vinyl 409 1 Allyl 410 1 CF₃ 411 1

412 1

413 1

414 1

415 1

416 1

417 1 H 418 1

419 1

420 1

421 1

422 1

423 1

424 1

425 1

426 2 Methyl 427 2 Ethyl 428 2 Isopropyl 429 2 Butyl 430 2 t-Butyl 431 2Propyl 432 2 Benzyl 433 2 Vinyl 434 2 Allyl 435 2 CF₃ 436 2

437 2

438 2

439 2

440 2

441 2

442 2 H 443 2

444 2

445 2

446 2

447 2

448 2

449 2

450 2

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 451 to 525 in Table 7) accordingto Formula IV-C, wherein, R₁ and m are delineated for each compound inTable 7.

TABLE 7 Compound m R₁ 451 0 Methyl 452 0 Ethyl 453 0 Isopropyl 454 0Butyl 455 0 t-Butyl 456 0 Propyl 457 0 Benzyl 458 0 Vinyl 459 0 Allyl460 0 CF₃ 461 0

462 0

463 0

464 0

465 0

466 0

467 0 NH₂ 468 0

469 0

470 0

471 0

472 0

473 0

474 0

475 0 F 476 1 Methyl 477 1 Ethyl 478 1 Isopropyl 479 1 Butyl 480 1t-Butyl 481 1 Propyl 482 1 Benzyl 483 1 Vinyl 484 1 Allyl 485 1 CF₃ 4861

487 1

488 1

489 1

490 1

491 1

492 1 NH₂ 493 1

494 1

495 1

496 1

497 1

498 1

499 1

500 1 F 501 2 Methyl 502 2 Ethyl 503 2 Isopropyl 504 2 Butyl 505 2t-Butyl 506 2 Propyl 507 2 Benzyl 508 2 Vinyl 509 2 Allyl 510 2 CF₃ 5112

512 2

513 2

514 2

515 2

516 2

517 2 NH₂ 518 2

519 2

520 2

521 2

522 2

523 2

524 2

525 2 F

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 526 to 600 in Table 8) accordingto Formula IV-D, wherein, R₁ and m are delineated for each compound inTable 8.

TABLE 8 Compound m R₁ 526 0 Methyl 527 0 Ethyl 528 0 Isopropyl 529 0Butyl 530 0 t-Butyl 531 0 Propyl 532 0 Benzyl 533 0 Vinyl 534 0 Allyl535 0 CF₃ 536 0

537 0

538 0

539 0

540 0

541 0

542 0 NH₂ 543 0

544 0

545 0

546 0

547 0

548 0

549 0

550 0 F 551 1 Methyl 552 1 Ethyl 553 1 Isopropyl 554 1 Butyl 555 1t-Butyl 556 1 Propyl 557 1 Benzyl 558 1 Vinyl 559 1 Allyl 560 1 CF₃ 5611

562 1

563 1

564 1

565 1

566 1

567 1 NH₂ 568 1

569 1

570 1

571 1

572 1

573 1

574 1

575 1 F 576 2 Methyl 577 2 Ethyl 578 2 Isopropyl 579 2 Butyl 580 2t-Butyl 581 2 Propyl 582 2 Benzyl 583 2 Vinyl 584 2 Allyl 585 2 CF₃ 5862

587 2

588 2

589 2

590 2

591 2

592 2 NH₂ 593 2

594 2

595 2

596 2

597 2

598 2

599 2

600 2 F

A seventh embodiment of the invention is a compound represented byFormula VII-A or VII-B or a pharmaceutically acceptable salt, solvate,hydrate, ester or prodrug thereof:

wherein R₁ and m are as previously defined in Formula I or I′.

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 601 to 675 in Table 9) accordingto Formula VII-A, wherein, R₁ and m are delineated for each compound inTable 9.

TABLE 9 Compound m R₁ 601 0 Methyl 602 0 Ethyl 603 0 Isopropyl 604 0Butyl 605 0 t-Butyl 606 0 Propyl 607 0 Benzyl 608 0 Vinyl 609 0 Allyl610 0 CF₃ 611 0

612 0

613 0

614 0

615 0

616 0

617 0 NH₂ 618 0

619 0

620 0

621 0

622 0

623 0

624 0

625 0 F 626 1 Methyl 627 1 Ethyl 628 1 Isopropyl 629 1 Butyl 630 1t-Butyl 631 1 Propyl 632 1 Benzyl 633 1 Vinyl 634 1 Allyl 635 1 CF₃ 6361

637 1

638 1

639 1

640 1

641 1

642 1 NH₂ 643 1

644 1

645 1

646 1

647 1

648 1

649 1

650 1 F 651 2 Methyl 652 2 Ethyl 653 2 Isopropyl 654 2 Butyl 655 2t-Butyl 656 2 Propyl 657 2 Benzyl 658 2 Vinyl 659 2 Allyl 660 2 CF₃ 6612

662 2

663 2

664 2

665 2

666 2

667 2 NH₂ 668 2

669 2

670 2

671 2

672 2

673 2

674 2

675 2 F

Representative compounds of the invention include, but are not limitedto, the following compounds (compounds 676 to 750 in Table 10) accordingto Formula VII-A, wherein, R₁ and m are delineated for each compound inTable 10.

TABLE 10 Compound m R₁₀ 676 0 Methyl 677 0 Ethyl 678 0 Isopropyl 679 0Butyl 680 0 t-Butyl 681 0 Propyl 682 0 Benzyl 683 0 Vinyl 684 0 Allyl685 0 CF₃ 686 0

687 0

688 0

689 0

690 0

691 0

692 0 H 693 0

694 0

695 0

696 0

697 0

698 0

699 0

700 0

701 1 Methyl 702 1 Ethyl 703 1 Isopropyl 704 1 Butyl 705 1 t-Butyl 706 1Propyl 707 1 Benzyl 708 1 Vinyl 709 1 Allyl 710 1 CF₃ 711 1

712 1

713 1

714 1

715 1

716 1

717 1 H 718 1

719 1

720 1

721 1

722 1

723 1

724 1

725 1

726 2 Methyl 727 2 Ethyl 728 2 Isopropyl 729 2 Butyl 730 2 t-Butyl 731 2Propyl 732 2 Benzyl 733 2 Vinyl 734 2 Allyl 735 2 CF₃ 736 2

737 2

738 2

739 2

740 2

741 2

742 2 H 743 2

744 2

745 2

746 2

747 2

748 2

749 2

750 2

In certain embodiments, the present invention provides a method for theprevention or treatment of an FXR mediated disease or condition. Themethod comprises administering a therapeutically effective amount of acompound of the invention. The present invention also provides the useof a compound of the invention for the preparation of a medicament forthe prevention or treatment of an FXR mediated disease or condition.

In certain embodiments, the FXR-mediated disease or condition iscardiovascular disease, atherosclerosis, arteriosclerosis,hypercholesteremia, or hyperlipidemia chronic liver disease,gastrointestinal disease, renal disease, metabolic disease, cancer(i.e., colorectal cancer), or neurological indications such as stroke.

In certain embodiments, the chronic liver disease is primary biliarycirrhosis (PBC), cerebrotendinous xanthomatosis (CTX), primarysclerosing cholangitis (PSC), drug induced cholestasis, intrahepaticcholestasis of pregnancy, parenteral nutrition associated cholestasis(PNAC), bacterial overgrowth or sepsis associated cholestasis,autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease,nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis(NASH), liver transplant associated graft versus host disease, livingdonor transplant liver regeneration, congenital hepatic fibrosis,choledocholithiasis, granulomatous liver disease, intra- or extrahepaticmalignancy, Sjogren's syndrome, Sarcoidosis, Wilson's disease, Gaucher'sdisease, hemochromatosis, or alpha 1-antitrypsin deficiency. In certainembodiments, the gastrointestinal disease is inflammatory bowel disease(IBD) (including Crohn's disease and ulcerative colitis), irritablebowel syndrome (MS), bacterial overgrowth, malabsorption, post-radiationcolitis, or microscopic colitis.

In certain embodiments, the renal disease is diabetic nephropathy, focalsegmental glomerulosclerosis (FSGS), hypertensive nephrosclerosis,chronic glomerulonephritis, chronic transplant glomerulopathy, chronicinterstitial nephritis, or polycystic kidney disease.

In certain embodiments, the cardiovascular disease is atherosclerosis,arteriosclerosis, dyslipidemia, hypercholesterolemia, orhypertriglyceridemia.

In certain embodiments, the metabolic disease is insulin resistance,Type I and Type II diabetes, or obesity.

In yet another embodiment, the invention provides the use of thecompound or pharmaceutical composition of the invention, in themanufacture of a medicament for a treating or preventing a disease in asubject that involves modulation of the TGR5 receptor. The inventionincludes a method of treating or preventing a disease that involvesmodulation of the TGR5 receptor in a subject by administering a compoundor pharmaceutical composition of the invention.

In certain embodiments, a disease that involves modulation of the TGR5receptor is selected from metabolic disease, inflammatory disease, liverdisease, autoimmune disease, cardiac disease, kidney disease, cancer,and gastrointestinal disease.

In one aspect, the invention provides for the use, wherein the diseaseis an inflammatory disease selected from allergy, osteoarthritis,appendicitis, bronchial asthma, pancreatitis, allergic rash, andpsoriasis. The invention includes a method of treating or preventing aninflammatory disease selected from allergy, osteoarthritis,appendicitis, bronchial asthma, pancreatitis, allergic rash, andpsoriasis.

In one aspect, the invention provides for the use, wherein the diseaseis an autoimmune disease selected from rheumatoid arthritis, multiplesclerosis, and type I diabetes. The invention includes a method oftreating or preventing an autoimmune disease selected from rheumatoidarthritis, multiple sclerosis, and type I diabetes.

In one aspect, the invention provides for the use, wherein the diseaseis a gastrointestinal disease selected from inflammatory bowel disease(Crohn's disease, ulcerative colitis), short bowel syndrome(post-radiation colitis), microscopic colitis, irritable bowel syndrome(malabsorption), and bacterial overgrowth. The invention includes amethod of treating or preventing a gastrointestinal disease selectedfrom inflammatory bowel disease (Crohn's disease, ulcerative colitis),short bowel syndrome (post-radiation colitis), microscopic colitis,irritable bowel syndrome (malabsorption), and bacterial overgrowth.

In one aspect, the invention provides for the use, wherein the diseaseis kidney disease selected from diabetic nephropathy, chronic renalfailure, hypertensive nephrosclerosis, chronic glomerulonephritis,chronic transplant glomerulopathy, chronic interstitial nephritis, andpolycystic kidney disease. The invention includes a method of treatingor preventing kidney disease selected from diabetic nephropathy, chronicrenal failure, hypertensive nephrosclerosis, chronic glomerulonephritis,chronic transplant glomerulopathy, chronic interstitial nephritis, andpolycystic kidney disease.

In one aspect, the invention provides for the use, wherein the diseaseis cancer selected from colorectal cancer, liver cancer, hepatocellularcarcinoma, cholangio carcinoma, renal cancer, gastric cancer, pancreaticcancer, prostate cancer, and insulanoma. The invention includes a methodof treating or preventing cancer selected from colorectal cancer, livercancer, hepatocellular carcinoma, cholangio carcinoma, renal cancer,gastric cancer, pancreatic cancer, prostate cancer, and insulanoma.

In one aspect, the compound is a selective FXR agonist over TGR5activator.

In one aspect, the compound is a selective TGR5 agonist over FXRactivator.

In one aspect, the compound is a dual agonist for both FXR and TGR5.

Yet a further aspect of the present invention is a process of making anyof the compounds delineated herein employing any of the synthetic meansdelineated herein.

Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “alkyl”, as used herein, refers to a saturated, monovalentstraight- or branched-chain hydrocarbon group. Preferred alkyl radicalsinclude C₁-C₆ alkyl and C₁-C₈ alkyl radicals. Examples of C₁-C₆ alkylgroups include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl groups; and examplesof C₁-C₈ alkyl groups include, but are not limited to, methyl, ethyl,propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, andoctyl groups.

The term “alkenyl”, as used herein, denote a monovalent group derivedfrom a hydrocarbon moiety by the removal of a single hydrogen atomwherein the hydrocarbon moiety has at least one carbon-carbon doublebond. Preferred alkenyl groups include C₂-C₆ alkenyl and C₂-C₈ alkenylgroups. Alkenyl groups include, but are not limited to, for example,ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl andthe like.

The term “alkynyl”, as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety by the removal of a single hydrogen atomwherein the hydrocarbon moiety has at least one carbon-carbon triplebond. Preferred alkynyl groups include C₂-C₆ alkynyl and C₂-C₈ alkynylgroups. Representative alkynyl groups include, but are not limited to,for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl and thelike.

The term “carbocycle” refers to a saturated (e.g., “cycloalkyl”),partially saturated (e.g., “cycloalkenyl” or “cycloalkynyl”) orcompletely unsaturated (e.g., “aryl”) ring system containing zeroheteroatom ring atom. “Ring atoms” or “ring members” are the atoms boundtogether to form the ring or rings. Where a carbocycle group is adivalent moiety linking two other elements in a depicted chemicalstructure (such as Z in Formula IA), the carbocycle group can beattached to the two other elements through any two substitutable ringatoms. A C₄-C₆ carbocycle has 4-6 ring atoms.

The term “cycloalkyl”, as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated carbocyclic ringcompound by the removal of a single hydrogen atom. Preferred cycloalkylgroups include C₃-C₈ cycloalkyl and C₃-C₁₂ cycloalkyl groups. Examplesof C₃-C₈-cycloalkyl include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; andexamples of C₃-C₁₂-cycloalkyl include, but not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo[2.2.2] octyl.

The term “cycloalkenyl” as used herein, denote a monovalent groupderived from a monocyclic or polycyclic carbocyclic ring compound havingat least one carbon-carbon double bond by the removal of a singlehydrogen atom. Preferred cycloalkenyl groups include C₃-C₈ cycloalkenyland C₃-C₁₂ cycloalkenyl groups. Examples of C₃-C₈-cycloalkenyl include,but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like; and examples ofC₃-C₁₂-cycloalkenyl include, but not limited to, cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,and the like.

The term “aryl,” as used herein, refers to a mono- or bicycliccarbocyclic ring system having one or two aromatic rings including, butnot limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyland the like.

The term “arylalkyl,” as used herein, refers to a C₁-C₃ alkyl or C₁-C₆alkyl residue attached to an aryl ring. Examples include, but are notlimited to, benzyl, phenethyl and the like.

The term “heteroaryl,” as used herein, refers to a mono-, bi-, ortri-cyclic aromatic radical or ring having from five to ten ring atomsof which at least one ring atom is selected from S, O and N; wherein anyN or S contained within the ring may be optionally oxidized. Preferredheteroaryl groups are monocyclic or bicyclic. Heteroaryl groups include,but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

The term “heteroarylalkyl,” as used herein, refers to a C₁-C₃ alkyl orC₁-C₆ alkyl residue residue attached to a heteroaryl ring. Examplesinclude, but are not limited to, pyridinylmethyl, pyrimidinylethyl andthe like.

The term “substituted” as used herein, refers to independent replacementof one, two, or three or more of the hydrogen atoms thereon withsubstituents including, but not limited to, deuterium, —F, —Cl, —Br, —I,—OH, protected hydroxy, —NO₂, —CN, —NH₂, N₃, protected amino, alkoxy,thioalkoxy, oxo, -halo-C₁-C₁₂-alkyl, -halo-C₂-C₁₂-alkenyl,-halo-C₂-C₁₂-alkynyl, -halo-C₃-C₁₂-cycloalkyl, —NH—C₁-C₁₂-alkyl,—NH—C₂-C₁₂-alkenyl, —NH—C₂-C₁₂-alkynyl, —NH—C₃-C₁₂-cycloalkyl, —NH-aryl,—NH-heteroaryl, —NH-heterocycloalkyl, -dialkylamino, -diarylamino,-diheteroarylamino, —O—C₁-C₁₂-alkyl, —O—C₂-C₁₂-alkenyl,—O—C₂-C₁₂-alkynyl, —O—C₃-C₁₂-cycloalkyl, —O-aryl, —O-heteroaryl, —O—heterocycloalkyl, —C(O)—C₁-C₁₂-alkyl, —C(O)—C₂-C₁₂-alkenyl,—C(O)—C₂-C₁₂-alkynyl, —C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl,—C(O)-heteroaryl, —C(O)-heterocycloalkyl, —CONH₂, —CONH—C₁-C₁₂-alkyl,—CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkynyl, —CONH—C₃-C₁₂-cycloalkyl,—CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl,—OCO₂—C₁-C₁₂-alkyl, —OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₂-C₁₂-alkynyl,—OCO₂—C₃-C₁₂-cycloalkyl, —OCO₂-aryl, —OCO₂-heteroaryl,—OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,—OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkynyl, —OCONH—C₃-C₁₂-cycloalkyl,—OCONH-aryl, —OCONH-heteroaryl, —OCONH-heterocycloalkyl,—NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkynyl,—NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂—C₂-C₁₂-alkenyl,—NHCO₂—C₂-C₁₂-alkynyl, —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂-aryl,—NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,—NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkynyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂,—NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkynyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,—NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkynyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,—NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkynyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl,—C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkynyl,—C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl, —S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl,—S(O)—C₂-C₁₂-alkynyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl —SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkynyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkynyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl,-heteroaryl, -heteroarylalkyl, -heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH,—S—C₁-C₁₂-alkyl, —S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkynyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S-heterocycloalkyl,methylthiomethyl, or -L′-R′, wherein L′ is C₁-C₆alkylene,C₂-C₆alkenylene or C₂-C₆alkynylene, and R′ is aryl, heteroaryl,heterocyclic, C₃-C₁₂cycloalkyl or C₃-C₁₂cycloalkenyl. It is understoodthat the aryls, heteroaryls, alkyls, and the like can be furthersubstituted. In some cases, each substituent in a substituted moiety isadditionally optionally substituted with one or more groups, each groupbeing independently selected from —F, —Cl, —Br, —I, —OH, —NO₂, —CN, or—NH₂.

In accordance with the invention, any of the aryls, substituted aryls,heteroaryls and substituted heteroaryls described herein, can be anyaromatic group. Aromatic groups can be substituted or unsubstituted.

It is understood that any alkyl, alkenyl, alkynyl, cycloalkyl andcycloalkenyl moiety described herein can also be an aliphatic group, analicyclic group or a heterocyclic group. An “aliphatic group” isnon-aromatic moiety that may contain any combination of carbon atoms,hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, andoptionally contain one or more units of unsaturation, e.g., doubleand/or triple bonds. An aliphatic group may be straight chained,branched or cyclic and preferably contains between about 1 and about 24carbon atoms, more typically between about 1 and about 12 carbon atoms.In addition to aliphatic hydrocarbon groups, aliphatic groups include,for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines,and polyimines, for example. Such aliphatic groups may be furthersubstituted. It is understood that aliphatic groups may be used in placeof the alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylenegroups described herein.

The term “alicyclic,” as used herein, denotes a monovalent group derivedfrom a monocyclic or polycyclic saturated carbocyclic ring compound bythe removal of a single hydrogen atom. Examples include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1] heptyl, and bicyclo [2.2.2] octyl. Such alicyclic groups may befurther substituted.

The term “heterocycloalkyl” and “heterocyclic” can be usedinterchangeably and refer to a non-aromatic 3-, 4-, 5-, 6- or 7-memberedring or a bi- or tri-cyclic group fused system, where: (i) each ringcontains between one and three heteroatoms independently selected fromoxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 doublebonds and each 6-membered ring has 0 to 2 double bonds, (iii) thenitrogen and sulfur heteroatoms may optionally be oxidized, (iv) thenitrogen heteroatom may optionally be quaternized, (v) any of the aboverings may be fused to a benzene ring, and (vi) the remaining ring atomsare carbon atoms which may be optionally oxo-substituted. Representativeheterocycloalkyl groups include, but are not limited to, [1,3]dioxolane,pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, andtetrahydrofuryl. Such heterocyclic groups may be further substituted togive substituted heterocyclic.

It will be apparent that in various embodiments of the invention, thesubstituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, arylalkyl, heteroarylalkyl, andheterocycloalkyl are intended to be monovalent or divalent. Thus,alkylene, alkenylene, and alkynylene, cycloaklylene, cycloalkenylene,cycloalkynylene, arylalkylene, hetoerarylalkylene andheterocycloalkylene groups are to be included in the above definitions,and are applicable to provide the formulas herein with proper valency.

The term “hydroxy activating group”, as used herein, refers to a labilechemical moiety which is known in the art to activate a hydroxy group sothat it will depart during synthetic procedures such as in asubstitution or elimination reactions. Examples of hydroxy activatinggroup include, but not limited to, mesylate, tosylate, triflate,p-nitrobenzoate, phosphonate and the like.

The term “activated hydroxy”, as used herein, refers to a hydroxy groupactivated with a hydroxy activating group, as defined above, includingmesylate, tosylate, triflate, p-nitrobenzoate, phosphonate groups, forexample.

The term “protected hydroxy,” as used herein, refers to a hydroxy groupprotected with a hydroxy protecting group, as defined above, includingbenzoyl, acetyl, trimethylsilyl, triethylsilyl, and methoxymethylgroups.

The term “hydroxy protecting group,” as used herein, refers to a labilechemical moiety which is known in the art to protect a hydroxy groupagainst undesired reactions during synthetic procedures. After saidsynthetic procedure(s) the hydroxy protecting group as described hereinmay be selectively removed. Hydroxy protecting groups as known in theare described generally in T. H. Greene and P. G., S. M. Wuts,Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons,New York (1999). Examples of hydroxy protecting groups includebenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl,isopropoxycarbonyl, diphenylmethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl,2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl,trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl,2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, 1,1-dimethyl-2-propenyl,3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl,triphenylmethyl (trityl), tetrahydrofuryl, methoxymethyl,methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl,2-(trimethyl silyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl,trimethylsilyl, triethylsilyl, triisopropylsilyl, and the like.Preferred hydroxy protecting groups for the present invention are acetyl(Ac or —C(O)CH₃), benzoyl (Bz or —C(O)C₆H₅), and trimethylsilyl (TMS or—Si(CH₃)₃).

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids.The present invention is meant to include all such possible isomers, aswell as their racemic and optically pure forms. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques, which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Whenthe compounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included. Theconfiguration of any carbon-carbon double bond appearing herein isselected for convenience only and is not intended to designate aparticular configuration unless the text so states; thus a carbon-carbondouble bond depicted arbitrarily herein as trans may be cis, trans, or amixture of the two in any proportion.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art.

Berge, et al. describes pharmaceutically acceptable salts in detail inJ. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be preparedin situ during the final isolation and purification of the compounds ofthe invention, or separately by reacting the free base function with asuitable organic acid. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, nontoxic acid addition salts e.g.,salts of an amino group formed with inorganic acids such as hydrochloricacid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloricacid or with organic acids such as acetic acid, maleic acid, tartaricacid, citric acid, succinic acid or malonic acid or by using othermethods used in the art such as ion exchange. Other pharmaceuticallyacceptable salts include, but are not limited to, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

Pharmaceutically acceptable salts can also be prepared by deprotonationof the parent compound with a suitable base, thereby forming the anionicconjugate base of the parent compound. In such salts the counter ion isa cation. Suitable cations include ammonium and metal cations, such asalkali metal cations, including Li⁺, Na⁺, K⁺ and Cs⁺, and alkaline earthmetal cations, such as Mg²⁺ and Ca²⁺.

The term “amino protecting group,” as used herein, refers to a labilechemical moiety which is known in the art to protect an amino groupagainst undesired reactions during synthetic procedures. After saidsynthetic procedure(s) the amino protecting group as described hereinmay be selectively removed. Amino protecting groups as known in the aredescribed generally in T. H. Greene and P. G. M. Wuts, Protective Groupsin Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).Examples of amino protecting groups include, but are not limited to,t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and thelike.

As used herein, the term “pharmaceutically acceptable ester” refers toesters of the compounds formed by the process of the present inventionwhich hydrolyze in vivo and include those that break down readily in thehuman body to leave the parent compound or a salt thereof. Suitableester groups include, for example, those derived from pharmaceuticallyacceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Examples ofparticular esters include, but are not limited to, formates, acetates,propionates, butyrates, acrylates and ethyl succinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the present invention. “Prodrug”, as used hereinmeans a compound, which is convertible in vivo by metabolic means (e.g.by hydrolysis) to afford any compound delineated by the formulae of theinstant invention. Various forms of prodrugs are known in the art, forexample, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier(1985); Widder, et al. (ed.), Methods in Enzymology, Vol. 4, AcademicPress (1985); Krogsgaard-Larsen, et al., (ed). “Design and Applicationof Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191(1991); Bundgaard, et al., Journal of Drug Deliver Reviews,8:1-38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq.(1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug DeliverySystems, American Chemical Society (1975); and Bernard Testa & JoachimMayer, “Hydrolysis In Drug And Prodrug Metabolism: Chemistry,Biochemistry And Enzymology,” John Wiley and Sons, Ltd. (2002).

The term “treating”, as used herein, means relieving, lessening,reducing, eliminating, modulating, or ameliorating, i.e. causingregression of the disease state or condition. Treating can also includeinhibiting, i.e. arresting the development, of a existing disease stateor condition, and relieving or ameliorating, i.e. causing regression ofan existing disease state or condition, for example when the diseasestate or condition may already be present.

The term “preventing”, as used herein means, to completely or almostcompletely stop a disease state or condition, from occurring in apatient or subject, especially when the patient or subject ispredisposed to such or at risk of contracting a disease state orcondition.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar to or comparable in function and appearance tothe reference compound.

The term “aprotic solvent,” as used herein, refers to a solvent that isrelatively inert to proton activity, i.e., not acting as a proton-donor.Examples include, but are not limited to, hydrocarbons, such as hexaneand toluene, for example, halogenated hydrocarbons, such as, forexample, methylene chloride, ethylene chloride, chloroform, and thelike, heterocyclic compounds, such as, for example, tetrahydrofuran andN-methylpyrrolidinone, and ethers such as diethyl ether,bis-methoxymethyl ether. Such solvents are well known to those skilledin the art, and individual solvents or mixtures thereof may be preferredfor specific compounds and reaction conditions, depending upon suchfactors as the solubility of reagents, reactivity of reagents andpreferred temperature ranges, for example. Further discussions ofaprotic solvents may be found in organic chemistry textbooks or inspecialized monographs, for example: Organic Solvents PhysicalProperties and Methods of Purification, 4th ed., edited by John A.Riddick et al., Vol. II, in the Techniques of Chemistry Series, JohnWiley & Sons, N Y, 1986.

The terms “protogenic organic solvent” or “protic solvent” as usedherein, refer to a solvent that tends to provide protons, such as analcohol, for example, methanol, ethanol, propanol, isopropanol, butanol,t-butanol, and the like. Such solvents are well known to those skilledin the art, and individual solvents or mixtures thereof may be preferredfor specific compounds and reaction conditions, depending upon suchfactors as the solubility of reagents, reactivity of reagents andpreferred temperature ranges, for example. Further discussions ofprotogenic solvents may be found in organic chemistry textbooks or inspecialized monographs, for example: Organic Solvents PhysicalProperties and Methods of Purification, 4th ed., edited by John A.Riddick et al., Vol. II, in the Techniques of Chemistry Series, JohnWiley & Sons, N Y, 1986.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject).

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. Additionally, thevarious synthetic steps may be performed in an alternate sequence ororder to give the desired compounds. In addition, the solvents,temperatures, reaction durations, etc. delineated herein are forpurposes of illustration only and variation of the reaction conditionscan produce the desired bridged macrocyclic products of the presentinvention. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein include, for example, those described in R.Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995).

The compounds of this invention may be modified by appending variousfunctionalities via synthetic means delineated herein to enhanceselective biological properties. Such modifications include those whichincrease biological penetration into a given biological system (e.g.,blood, lymphatic system, central nervous system), increase oralavailability, increase solubility to allow administration by injection,alter metabolism and alter rate of excretion.

Pharmaceutical Compositions

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers. As used herein, the term “pharmaceutically acceptable carrier”means a non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are sugars such as lactose, glucose and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil;safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols;such a propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and otheranimals orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,or drops), buccally, or as an oral or nasal spray.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1, 3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or: a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

Unless otherwise defined, all technical and scientific terms used hereinare accorded the meaning commonly known to one with ordinary skill inthe art. All publications, patents, published patent applications, andother references mentioned herein are hereby incorporated by referencein their entirety.

Abbreviations

Abbreviations which have been used in the descriptions of the schemesand the examples that follow are:

-   -   ACN for acetonitrile;    -   BME for 2-mercaptoethanol;    -   BOP for benzotriazol-1-yloxy-tris(dimethylamino)phosphonium        hexafluorophosphate;    -   BzCl for benzoyl chloride;    -   CDI for carbonyldiimidazole;    -   COD for cyclooctadiene;    -   DABCO for 1,4-diazabicyclo[2.2.2]octane;    -   DAST for diethylaminosulfur trifluoride;    -   DABCYL for        6-(N-4′-carboxy-4-(dimethylamino)azobenzene)-aminohexyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite;    -   DBU for 1, 8-Diazabicycloundec-7-ene;    -   DCC for N, N′-dicyclohexylcarbodiimide;    -   DCM for dichloromethane;    -   DIAD for diisopropyl azodicarboxylate;    -   DIBAL-H for diisobutylaluminum hydride;    -   DIPEA for diisopropyl ethylamine;    -   DMAP for N,N-dimethylaminopyridine;    -   DME for ethylene glycol dimethyl ether;    -   DMEM for Dulbecco's Modified Eagles Media;    -   DMF for N,N-dimethyl formamide;    -   DMSO for dimethylsulfoxide;    -   DSC for N, N′-disuccinimidyl carbonate;    -   DPPA for diphenylphosphoryl azide;    -   DUPHOS for

-   -   EDANS for 5-(2-Amino-ethylamino)-naphthalene-1-sulfonic acid;    -   EDCI or EDC for 1-(3-diethylaminopropyl)-3-ethylcarbodiimide        hydrochloride;    -   EtOAc for ethyl acetate;    -   EtOH for ethyl alcohol;    -   HATU for O        (7-Azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate;    -   HCl for hydrochloric acid;    -   Hoveyda's Cat. for Dichloro(o-isopropoxyphenylmethylene)        (tricyclohexylphosphine)ruthenium(II);

In for indium;

-   -   KHMDS is potassium bis(trimethylsilyl) amide;    -   Ms for mesyl;    -   NMM for N-4-methylmorpholine;    -   NMI for N-methylimidazole;    -   NMO for N-4-methylmorpholine-N-Oxide;    -   PyBrOP for Bromo-tri-pyrolidino-phosphonium hexafluorophosphate;    -   Ph for phenyl;    -   RCM for ring-closing metathesis;    -   RT for reverse transcription;    -   RT-PCR for reverse transcription-polymerase chain reaction;    -   TBME for tert-butyl methyl ether;    -   TEA for triethyl amine;    -   Tf₂O for trifluoromethanesulfonic anhydride;    -   TFA for trifluoroacetic acid;    -   THF for tetrahydrofuran;    -   TLC for thin layer chromatography;    -   (TMS)₂NH for hexamethyldisilazane;    -   TMSOTf for trimethylsilyl trifluoromethanesulfonate;    -   TBS for t-Butyldimethylsilyl;    -   TMS for trimethylsilyl;    -   TPAP tetrapropylammonium perruthenate;    -   TPP or PPh₃ for triphenylphosphine;    -   TrCl for trityl chloride;    -   DMTrCl for 4,4′-dimethoxytrityl chloride;    -   tBOC or Boc for tert-butyloxy carbonyl.        Synthetic Methods

The compounds and processes of the present invention will be betterunderstood in connection with the following synthetic schemes thatillustrate the methods by which the compounds of the invention may beprepared, which are intended as an illustration only and not to limitthe scope of the invention. Various changes and modifications to thedisclosed embodiments will be apparent to those skilled in the art andsuch changes and modifications including, without limitation, thoserelating to the chemical structures, substituents, derivatives, and/ormethods of the invention may be made without departing from the spiritof the invention and the scope of the appended claims.

As shown in Scheme 1, novel bile acid analogs of the compound of formula(1-5) are prepared from the compound of formula (1-1), wherein R₁, m,and R₇ are defined as previously, P₁ and P₂ are hydroxyl protectinggroups. Thus, the two hydroxyl groups of the compound of formula (1-1)are protected with P₁ and P₂ groups to afford the compound of formula(1-2). P₁ and P₂ can be same or different. P₁ and P₂ can be any hydroxylprotecting group such as, but not limited to Ac, Bz, chloroacetyl, TES,TBS, MOM and Bn. A more detailed discussion of the procedures, reagentsand conditions for protection of hydroxyl group is described inliterature, for example, by T. W. Greene and P. G. M. Wuts in“Protective Groups in Organic Synthesis” 3^(rd) ed., John Wiley & Son,Inc., 1999. Then, the compound of formula (1-2) is converted to the acylazide compound of formula (1-3) using suitable reagent such as, but notlimited to, DPPA. The reaction solvent can be, but not limited to, THF,DCM and toluene. The preferred solvent is THF. The reaction temperatureis from −20° C.˜40° C. Further rearrangement of the compound of formula(1-3) at elevated temperature and reacting with sulfonamide afford thecompound of formula (1-4). Then deprotection of P₁ and P₂ groups affordthe sulfonylurea compound of formula (1-5). A more detailed discussionof the procedures, reagents and conditions for deprotection of hydroxylpretecting groups is described in literature, for example, by T. W.Greene and P. G. M. Wuts in “Protective Groups in Organic Synthesis”3^(rd) ed., John Wiley & Son, Inc., 1999.

Scheme 2 illustrates the preparation of the urea compound of formula(2-2) from the compound of formula (1-3), wherein R₁₀, m, and R₇ aredefined as previously, P₁ and P₂ are hydroxyl protecting groups. Thus,rearrangement of the compound of formula (1-3) at elevated temperatureand reacting with amine afford the compound of formula (2-1). Thendeprotection of P₁ and P₂ groups afford the urea compound of formula(2-2). A more detailed discussion of the procedures, reagents andconditions for deprotection of hydroxyl pretecting groups is describedin literature, for example, by T. W. Greene and P. G. M. Wuts in“Protective Groups in Organic Synthesis” 3^(rd) ed., John Wiley & Son,Inc., 1999.

Scheme 3 illustrates the preparation of the sulfonamide compound offormula (3-4) from the compound of formula (1-3), wherein R₁, m and R₇are defined as previously, P₁ and P₂ are hydroxyl protecting groups.Thus, the compound of formula (1-3) is converted to the compound offormula (3-1) through Curtius rearrangement. A more detailed discussionof the procedures, reagents and conditions for Curtius rearrangement isdescribed in literature, for example, by Jerry March in “AdvancedOrganic Chemistry” 4^(th) ed., John Wiley & Son, Inc., 1992. Then Bocdeprotection of the compound of formula (3-1) in acidic condition affordamine compound of formula (3-2). Then the compound of formula (3-2)reacts with sulfonyl chloride to give the sulfonamide compound offormula (3-3). Further deprotection of hydroxyl protecting group P₁ andP₂ to give the compound of formula (3-4). A more detailed discussion ofthe procedures, reagents and conditions for protection and deprotectionof hydroxyl pretecting groups and amino protecting group are describedin literature, for example, by T. W. Greene and P. G. M. Wuts in“Protective Groups in Organic Synthesis” 3^(rd) ed., John Wiley & Son,Inc., 1999.

An alternative procedure to prepare sulfonamide compound of formula(4-3) is illustrated in scheme 4, wherein R₁, m and R₇ are defined aspreviously, P₁ and P₂ are hydroxyl protecting groups. The compound offormula (1-2) is coupled with sulfonamide using suitable couplingcondition to give the compound of formula (4-1). The coupling reagentcan be selected from, but not limited to, DCC, EDC, CDI, di-isopropylcarbodiimide, BOP-Cl, PyBOP, PyAOP, TFFH and HATU. Suitable basesinclude, but are not limited to, triethylamine, diisopropylethylamine,DBU, N-methylmorpholine and DMAP. The coupling reaction is carried outin an aprotic solvent such as, but not limited to, CH₂C₁₂, DMF or THF.The reaction temperature can vary from 0° C. to about 50° C. Thecompound of formula (4-1) is treated with reducing agent to give thecompound of formula (4-2). The reducing agent can be selected from, butnot limited to LiAlH₄, LiBH₄, DIBAL, BH₃. The reaction is carried out inaprotic solvent such as, but not limited to, CH₂C₁₂, DMF or THF. Thereaction temperature can vary from 0° C. to about 100° C. Further,deprotection of the compound of formula of (4-2) give the compound offormula (4-3). A more detailed discussion of the procedures, reagentsand conditions for deprotection of hydroxyl pretecting groups isdescribed in literature, for example, by T. W. Greene and P. G. M. Wutsin “Protective Groups in Organic Synthesis” 3^(rd) ed., John Wiley &Son, Inc., 1999.

EXAMPLES

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

Example 1

Step 1-1:

To a 1-dram vial were added 6(α)-ethyl-chenodeoxycholic acid (6-ECDCA)(100 mg, 0.24 mmol), anhydrous THF (3 mL), TBSCl (107.5 mg, 0.71 mmol),and imidazole (57.2 mg, 0.84 mmol) respectively and the reaction mixturewas stirred at rt for 22 h. Transferred to a separation funnel, dilutedwith EtOAC (50 mL) and washed with brine-water (10 mL, 1:1 v/v). Dried,filtered and concentrated and the resulting white solid was dissolved inMeOH (10 mL) and was added K₂CO₃ (49.7 mg, 0.36 mmol). The mixture wasstirred at room temperature for 30 min. and cooled to 0° C. Then thereaction mixture was acidified by addition of 0.1 NHCl to PH<7 anddiluted with EtOAc (30 mL). After washed with brine (10 mL), dried, andconcentrated, the resulting crude material purified by CombiFlash (12 gSiO₂, Acetone/Hexanes=0˜40%) to give the compound (1) as a white solid,88 mg, 65.6% yield over 2 steps.

Step 1-2:

The TBS protected 6(α)-ethyl-chenodeoxycholic acid compound (1) (125 mg,0.23 mmol) was dissolved in THF (2.0 mL) and cooled to 0° C. To thesolution was added Et₃N (64 μL, 0.46 mmol) and diphenylphosphoryl azide(74 μL, 0.35 mmol). The mixture was stirred at 0° C. for 1.5 h, quenchedwith brine and extracted with DCM (2×). The combined organic layer wasdried over Na₂SO₄, filtered and concentrated in vacuo at 25° C. Theresidue was taken up in hexane, filtered through Na₂SO₄ and concentratedin vacuo at 25° C. The crude product compound (2) (150 mg) was used fornext step without purification.

Step 1-3:

The acyl azide compound (2) obtained above (75 mg) was dissolved intoluene (2.5 mL) and refluxed for 5 hrs. The mixture was cooled to roomtemperature and added naphthalene-2-sulfonamide (58 mg, 0.24 mmol) andDBU (36 μL, 0.24 mmol). The reaction mixture was stirred at roomtemperature for 1 hr, quenched with aq. 1 M HCl, and extracted withEtOAc (2×). The combined organic layer was dried over Na₂SO₄, filteredand concentrated in vacuo. The residue was purified by SiO₂chromatography with 0-50% EtOAc/hexane as eluent to provide N-sulfonylurea compound (3) (74 mg).

Step 1-4:

The above obtained compound (3) (74 mg) was dissolved in MeOH (1.0 mL)followed by addition of 1 drop of 37% conc. HCl. The mixture was stirredat room temperature for 10 min, quenched with sat. NaHCO₃, and extractedwith EtOAc (3×). The combined organic layer was dried over Na₂SO₄,filtered and concentrated in vacuo. The residue was by SiO₂chromatography with 0-50% acetone/hexane as eluent to provide thecompound of example 1 (44 mg).

The examples from example 2 to example 8 were prepared using sameprocedure as the one used in example 1. The MS data and 1H NMR data aredelineated in Table 9.

TABLE 9 Example R ESMS: ¹H NMR (500 MHz) 1

623.35 (M − 1) 2

657.25 (M − 1) 7.95 (2H, d, J = 7.5 Hz), 7.37 (2H, d, J = 7.5 Hz), 6.42(1H, br s), 3.70 (1H, br s), 3.41 (1H, br s), 3.30 (1H, br s), 3.17 (1H,br s), 0.94 (3H, d, J = 7.0 Hz), 0.89 (6H, m), 0.64 (3H, s). 3

629.34 (M − 1) 7.80 (2H, d, J = 7.5 Hz), 7.55 (2H, d, J = 7.5 Hz), 7.40(1H, br s), 6.55 (1H, br s), 3.71 (1H, br s), 3.41 (1H, br s), 3.30 (1H,br s), 3.18 (1H, m), 1.35 (9H, s), 0.95 (3H, d, J = 6.0 Hz), 0.92 (3H,m), 0.90 (3H, s), 0.66 (3H, s). 4

573.28 (M − 1) 5

537.28 (M − 1) 6

511.26 (M − 1) 7 iPr 477.40 3.85 (1H, br s), 3.70 (1H, br s), 3.41(M + 1) (1H, br s), 3.23 (1H, m), 3.12 (1H, m), 1.96 (1H, d, J = 12 Hz),1.22 (6H, d, J = 6.0 Hz), 0.98 (3H, d, J = 5.5 Hz), 0.90 (6H, br s),0.67 (3H, s).

Example 8

Step 8-1:

6(α)-ethyl-chenodeoxycholic acid (2.1 g, 2.38 mmol) was dissolved intoluene (11 ml). To the solution was added formic acid (98%, 3.0 mL) andperchloric acid (70%, 20 μL) dropwisely. The mixture was stirred at 105°C. for 3.5 h and cooled to room temperature. The mixture was dilutedwith EtOAc and washed with brine. The organic layer was dried overNa₂SO₄ and concentrated in vacuo. Purification of the residue by SiO₂chromatography with 0-40% acetone/hexane provided compound (8-1) (730mg).

Step 8-2:

The compound (8-1) (730 mg, 1.53 mmol) was dissolved in THF (8.0 mL) andcooled to 0° C. To the solution was added Et₃N (425 μL, 3.06 mmol) anddiphenylphosphoryl azide (347 μL, 1.61 mmol). The mixture was stirred at0° C. for 1.5 h, quenched with water, and extracted with EtOAc (2×). Thecombined organic layer was dried over Na₂SO₄, filtered and concentratedin vacuo at 25° C. The crude obtained above was dissolved in toluene (20mL), stirred at 100° C. for 30 min and t-BuOH (1.5 mL) was added. Themixture was stirred at 100° C. for 18 h, cooled to room temperature,diluted with EtOAc, and washed with water and brine. The organic layerwas dried over Na₂SO₄, filtered and concentrated in vacuo. Purificationof the residue by SiO₂ chromatography using 0-20% EtOAc/hexane providedcompound (8-2) (401 mg). LC/MS Observed [M+NH₄]⁺, 565.42. ¹HNMR (500MHz, CDCl₃) 8.15 (1H, s), 8.04 (1H, s), 5.19 (1H, s), 4.71 (1H, br s),4.41 (1H, br s), 3.19 (1H, br s), 3.03 (1H, br s), 1.44 (9H, s), 0.95(6H, br s), 0.90 (3H, t, J=7.0 Hz), 0.65 (3H, s).

Step 8-3:

Compound (8-2) (401 mg, 0.73 mmol) was dissolved in DCM (15 mL) andcooled to 0° C. TFA (1.1 mL) was added dropwise and the reactionsolution was warmed to room temperature and stirred for 1 h. The solventwas removed in vacuo. The residue was dissolved in DCM and washed withsat. NaHCO₃. The organic layer was collected, dried over Na₂SO₄, andconcentrated in vacuo. Compound (8-3) (300 mg) was obtained as a whitesolid. LC/MS observed [M+H]⁺, 448.

Step 8-4:

The amine compound (8-3) (100 mg, 0.22 mmol) was dissolved in DCM (1.0mL), followed by addition of Et₃N (67 μL, 0.48 mmol) andphenylmethanesulfonyl chloride (46 mg, 0.24 mmol). The reaction mixturewas stirred at room temperature for 18 hrs, quenched with 5% NaHCO₃, andextracted with DCM (3×). The combined organic layers was dried overNa₂SO₄ and concentrated in vacuo. Purification of the residue by SiO₂chromatography using 0-30% EtOAc/hexane provided compound (8-4) (57 mg).LC/MS observed [M+NH₄]⁺, 619.38; [M+HCOOH−H]⁻, 646.27. ¹HNMR (500 MHz,CDCl₃) 8.15 (1H, s), 8.03 (1H, s), 7.38 (5H, s), 5.18 (1H, s), 4.70 (1H,br s), 4.24 (2H, s), 3.94 (1H, br s), 3.02 (1H, br s), 2.93 (1H, br s),0.95 (3H, s), 0.89 (6H, m), 0.63 (3H, s).

Step 8-5:

Compound (8-4) (57 mg, 0.095 mmol) was dissolved in MeOH (0.5 mL). Tothe solution was added aq. 50% NaOH solution (0.23 mL, 30 eq). Themixture was stirred at 50° C. for 15 h, cooled to room temperature,quenched with 1 M HCl, and extracted with EtOAc (3×). The organic layerwas dried over Na₂SO₄ and concentrated in vacuo. Purification of theresidue provided compound of example 8. LC/MS observed [M+HCOOH−H]⁻,590.25.

Example 9

Compound (8-3) (57 mg, 0.095 mmol) was dissolved in MeOH (0.5 mL). Tothe solution was added aq. 50% NaOH solution (0.23 mL, 30 eq). Themixture was stirred at 50° C. for 15 h, cooled to room temperature,quenched with 1 M HCl, and extracted with EtOAc (3×). The organic layerwas dried over Na₂SO₄ and concentrated in vacuo. Purification of theresidue provided compound of example 9. LC/MS observed [M+1], 392.25.

Example 10

The acylsulfonamide (10-1) (100 mg, 0.18 mmol) was dissolved in THF (2mL). To the solution at −78° C. was added LiAlH₄ solution in THF (1.0 Min THF, 1.44 mL) dropwise. The mixture was warmed to room temperatureand stirred for 1 h. The reaction was quenched at 0° C. with saturatedpotassium sodium tartrate and stirred at room temperature for 14 hrs.The mixture was extracted with EtOAc (2×) and the combined organic layerwas dried over Na₂SO₄ and concentrated in vacuo. Purification of theresidue by SiO₂ chromatography with 0-50% EtOAc/hexane provided compoundof example 10 (42 mg). LC/MS observed [M+HCOOH−H]⁻, 590.29. ¹HNMR (500MHz, CDCl₃) 7.87 (2H, d, J=7.5 Hz), 7.59 (1H, m), 7.52 (2H, m), 4.33(1H, br s), 3.69 (1H, s), 3.41 (1H, br s), 3.92 (2H, br s), 0.90 (3H, t,J=7.5 Hz), 0.89 (3H, s), 0.85 (3H, d, J=5.5 Hz), 0.62 (3H, s).

Example 11

The compound of example 11 was prepared using a similar procedure as thecompound of example 10. LC/MS observed [M+HCOOH−H]⁻, 556.31. ¹HNMR (500MHz, CDCl₃) 3.69 (1H, br s), 3.40 (1H, br s), 3.14 (1H, m), 3.09 (2H,m), 1.95 (1H, d, J=12 Hz), 1.37 (6H, d, J=5.5 Hz), 0.93-0.89 (9H, m),0.65 (3H, s).

The examples from example 129 to example 143 were prepared using similarprocedure as above. The MS data are delineated in Table 10.

TABLE 10 Example # Structure MS data 129

[M + HCOOH − 1]⁻, 646.42 130

[M + HCOOH − 1]⁻, 576.26 131

[M − 1]⁻, 470 132

[M − 2H₂O + 1]⁺, 478.4 133

[M + 1]⁺, 615.60 134

[M − 2H₂O + 1]⁺, 558.50 135

[M − 1]⁻, 484 136

[M − 1]⁻, 538.40 137

[M − 1]⁻, 558.35 138

[M − 2H₂O + 1]⁺, 506.35 139

[M − 1]⁻, 510.45 140

[M − 1]⁻, 512.30 141

[M − 2H₂O + 1]⁺, 492.50 142

[M − 1]⁻, 599.20 143

[M − 1]⁻, 496.2

Example 108

5-Phenylthiophene-2-sulfonamide (145 mg, 0.6 mmol) and DBU (91 mg, 0.6mmol) in THF (5 mL) was added into a solution of the compound (2a) (1mL, 0.2 mmol) in toluene. The mixture was stirred at RT for overnight.The mixture was quenched with water, extracted with ethyl acetate (50mL), dried, filtered, and concentrated. The residue was dissolved inMeOH (2 mL), then 1 drop of 37% HCl was added. The mixture was stirredat rt for 10 minutes, then was diluted with ethyl acetate (50 mL) andwashed sequentially with saturated sodium bicarbonate and brine. Theorganic layer was dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18;mobile phase, MeCN/H₂O, Detector, UV 254 nm) to give the example 108(15.5 mg) as a white solid.

Example 109

1). Synthesis of Compound (109-1)

Compound (109-SM) (1 g, 10 mmol) was added to chlorosulfonic acid (10mL) at room temperature and the mixture was slowly warmed to 100° C.,and then heated at 100-110° C. for 2 hours. The reaction mixture wascooled, poured into 150 mL of crushed ice with stirring, and extractedwith ethyl acetate (20 mL*3). The organic layer was combined, washedwith saturated brine (30 mL), dried over Na₂SO₄, evaporated to obtain1.25 g of the title compound (109-1) as a yellow oil which was used inthe next step directly.

2). Synthesis of Compound (109-2)

A solution of the compound (109-1) (1.25 g, 6.48 mmol) in THF (40 mL)and ammonia (40 mL) was stirred at rt for 1.5 h. Then the solution wasconcentrated. The residue was purified by flash silica chromatography,elution gradient 40 to 100% EtOAc in petroleum ether to give the titlecompound (109-2) (510 mg, 45%) as a yellow solid.

3). Synthesis of Example 109

Compound (109-2) (87.5 mg, 0.5 mmol) and DBU (76 mg, 0.5 mmol) in THF (5mL) was added into a solution of the compound (2a) (1 mL, 0.2 mmol) intoluene. The mixture was stirred at RT for overnight. The mixture wasquenched with water, extracted with ethyl acetate (50 mL), dried,filtered, and concentrated. The residue was dissolved in MeOH (2 mL),then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes, then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 220 nm) to give example 109 (25.1 mg) as a whitesolid.

Example 111

1). Synthesis of Compound (111-1)

A solution of thiophene (6.0 g, 71.31 mmol) and 2-bromo-2-methylpropane(9.7 g, 70.79 mmol) in DCM (60 mL) was added dropwise totrichloroalumane (9.4 g, 70.50 mmol) in DCM (60 mL) at −78° C. Theresulting solution was stirred at −78° C. for 2 h and warmed to rt forovernight. The resulting mixture was diluted with DCM (200 mL), andwashed with water (50 mL), 5% sodium hydroxide (50 mL) and saturatedbrine (50 mL) sequentially. The organic layer was dried over Na₂SO₄ andconcentrated under vacuum to give 6.1 g (crude) of desired compound as ayellow oil.

2). Synthesis of Compound (111-2)

A solution of (111-1) (5 g, 35.7 mmol) in DCM (10 mL) was added dropwiseto an ice-cold solution of chlorosulfonic acid (12.4 g, 107 mmol) in DCM(30 mL). The reaction mixture was stirred at 0° C. for 30 min, thenpoured into ice. The solution was extracted with DCM (20 mL*3). Thecombined organic layer was washed with H₂O (30 mL), and saturated NaCl(30 mL), then dried over Na₂SO₄, filtered and evaporated to give 4.1 g(crude) of (111-2) as a yellow oil which was used in the next stepdirectly.

3). Synthesis of Compound (111-3)

A solution of (111-2) (4.1 g, 17.2 mmol) in THF (40 mL) and ammonia (40mL) was stirred at rt for 1.5 hours. Then was concentrated. The residuewas purified by flash silica chromatography, elution gradient 40 to 100%EtOAc in petroleum ether to give the title compound (2.1 g, 56%) as ayellow solid.

4). Synthesis of Example 111

Compound (113-3) (110 mg, 0.5 mmol) and DBU (76 mg, 0.5 mmol) in THF (5mL) was added into a solution of the compound (2a) (1 mL, 0.2 mmol) inPhCH₃. The mixture was stirred at RT for overnight. The mixture wasquenched with water, extracted with ethyl acetate (50 mL), dried,filtered, and concentrated. The residue was dissolved in MeOH (2 mL),then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes, then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 254 nm) to give the example 111 (29.4 mg) as awhite solid.

Example 112

1). Synthesis of Compound (112-1)

A solution of Compound (112-SM) (4 mL) in DCM (10 mL) was added dropwiseto an ice-cold solution of chlorosulfonic acid (10 mL) in DCM (30 mL).The reaction mixture was stirred for 2 h at RT, then poured into ice.The solution was extracted with DCM (20 mL*3). The combined organiclayer was washed with saturated brine (30 mL), dried over Na₂SO₄,filtered, and evaporated to give 1.1 g (crude) of (112-1) as a yellowoil, which was used in the next step directly.

2). Synthesis of Compound (112-2)

A solution of the compound (112-1) (1.1 g) in THF (40 mL) and ammonia(40 mL) was stirred at rt for 1.5 h. Then the solution was concentrated.The residue was purified by flash silica chromatography, elutiongradient 50 to 100% EtOAc in petroleum ether to give 550 mg of titlecompound (112-2) as a yellow solid.

3). Synthesis of Example 112

Compound (112-2) (100 mg, 0.5 mmol) and DBU (76 mg, 0.5 mmol) in THF (5mL) was added into a solution of compound (2a) (1 mL, 0.2 mmol) intoluene. The mixture was stirred at RT for overnight. The mixture wasquenched with water, extracted with ethyl acetate (50 mL), dried,filtered, and concentrated. The residue was dissolved in MeOH (2 mL),then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes, then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 254 nm) to give example 112 (21.5 mg, 18%) as awhite solid.

Example 113

1). Synthesis of Compound (113-1)

Into a 1000 mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of 6-ECDCA (18.0 g, 0.04mol, 1.00 eq) in THF (200 mL), TEA (86.5 g, 0.86 mol, 20.0 eq),4-dimethylaminopyridine (0.63 g, 0.004 mol, 0.1 eq), and aceticanhydride (87.3 g, 0.86 mol, 20.0 eq). The resulting solution wasstirred at 90° C. for 15 hours. After being cooled to rt, it wasconcentrated and the residue was dissolved in ethyl acetate (500 mL),then was washed with water (100 mL*2), saturated NaCl (100 mL*2). Theorganic layer was dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by flash silica chromatography, elution gradient 0to 20% EtOAc in petroleum ether to give the desired compound (113-1) asa yellow solid (20.0 g, 92.6%).

2). Synthesis of Compound (113-2)

Into a solution of (93-1) (20.0 g, 40 mmol) in TFA (150 mL) was addedTFAA (63.0 g, 300 mmol). Then NaNO₂ was added in 5 portions over 45minutes at 0° C. After stirred at 0° C. for 1 hour, the solution wasmoved to 40° C. for 40 minutes. The solution was quenched with waterafter being cooled to rt, then extracted with ethyl acetate (200 mL*3).The organic layer was dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by flash silica chromatography, elution gradient 10to 30% EtOAc in petroleum ether to give the desired compound as a yellowsolid (12.2 g, 65.6%).

3). Synthesis of Compound (113-3)

Into a solution of (113-2) (11.7 g, 24.8 mmol) in CH₃OH (100 mL) wasadded KOH (50.0 g, 892.8 mmol) and H₂O (100 mL). The mixture was stirredat 90° C. for 16 hours. The reaction mixture was quenched with 6 N HClto adjust pH to 5˜6, extracted with ethyl acetate (200 mL*3). Theorganic layer was dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by flash silica chromatography, elution gradient 0to 10% MeOH in DCM to give the desired compound as a yellow solid (9.0g, 89%).

4). Synthesis of Compound (113-4)

Into a solution of (113-3) (5.0 g, 12.3 mmol) in THF (200 mL) was addedImidazole (5.9 g, 86.1 mmol) and TBSCl (5.6 g, 36.9 mmol). The mixturewas stirred at rt for 16 hours. The reaction mixture was quenched with10% citric acid solution to adjust pH to 5˜6, extracted with ethylacetate (150 mL*3). The organic layer was dried over Na₂SO₄, filteredand concentrated to give crude product (7.2 g, crude) as a yellow solid.To the yellow solid in CH₃OH (250 mL) was added K₂CO₃ (2.3 g, 17.0mmol). The mixture was stirred at rt for 4 hours. The reaction mixturewas quenched with 10% citric acid solution to adjust pH to 5˜6,extracted with ethyl acetate (150 mL*3). The organic layer was driedover Na₂SO₄, filtered and concentrated. The residue was purified byflash silica chromatography, elution gradient 10 to 20% EtOAc inpetroleum ether to give the desired compound as a yellow solid (3.3 g,56%).

5). Synthesis of Compound (113-5)

Into a solution of (113-4) (1.0 g, 2.0 mmol) in toluene (10 mL) wasadded TEA (4.2 mmol, 2.1 eq) and DPPA (2.1 mmol, 1.05 eq) sequentiallyat 0° C. The resulting mixture was stirred at 0° C. for 1 hour. Then waswarmed to 100° C. and stirred for 5 hours. After being cooled to rt, a0.2 M solution of compound (113-5) in toluene was obtained, which can bedivided into several portions for the next step reaction.

6). Synthesis of Example 113

Cyclohexylbenzenesulfonamide (72 mg, 0.3 mmol) and DBU (0.153 g, 1 mmol)in THF (1 mL) was added into a solution of PH-ETA-C-005-5 (1 mL, 0.2mmol) in toluene. The mixture was stirred at rt for overnight. Themixture was quenched with water, extracted with ethyl acetate (20 mL*3),dried over Na₂SO₄, filtered, and concentrated. The residue was dissolvedin MeOH (2 mL), Then 1 drop of 37% HCl was added. The mixture wasstirred at rt for 10 minutes. Then the mixture was diluted with ethylacetate (50 mL) and washed sequentially with saturated sodiumbicarbonate and brine. The organic layer was dried over Na₂SO₄, filteredand concentrated. The residue was purified by Flash-Prep-HPLC((IntelFlash-1): Column, C18; mobile layer, MeCN/H₂O, Detector, UV 254nm) to give the example 113 as a white solid (44.7 mg).

Example 114

Compound (114-2) (71.7 mg, 0.3 mmol) and DBU (0.153 g, 1 mmol) in THF (1mL) was added into a solution of (113-5) (1 mL, 0.2 mmol) in toluene.The mixture was stirred at rt overnight. The mixture was quenched withwater, extracted with ethyl acetate (50 mL), dried over Na₂SO₄,filtered, and concentrated. The residue was dissolved in MeOH (2 mL),Then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes. then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 254 nm) to give example 114 as a white solid(12.7 mg).

Example 115

Compound (115-2) (63.9 mg, 0.3 mmol) and DBU (0.153 g, 1 mmol) in THF (1mL) was added into a solution of (113-5) (1 mL, 0.2 mmol) in toluene.The mixture was stirred at rt overnight. The mixture was quenched withwater, extracted with ethyl acetate (50 mL), dried over Na₂SO₄,filtered, and concentrated. The residue was dissolved in MeOH (2 mL),then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes. Then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 254 nm) to give example 115 as a white solid(25.9 mg).

Example 116

Compound (116-2) (72.3 mg, 0.3 mmol) and DBU (0.153 g, 1 mmol) in THF (1mL) was added into a solution of (113-5) (1 mL, 0.2 mmol) in toluene.The mixture was stirred at rt overnight. The mixture was quenched withwater, extracted with ethyl acetate (50 mL), dried over Na₂SO₄,filtered, and concentrated. The residue was dissolved in MeOH (2 mL),then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes. Then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 254 nm) to give example 116 as a white solid(24.2 mg).

Example 117

Compound (117-2) (60.3 mg, 0.3 mmol) and DBU (0.153 g, 1 mmol) in THF (1mL) was added into a solution of (113-5) (1 mL, 0.2 mmol) in toluene.The mixture was stirred at rt overnight. The mixture was quenched withwater, extracted with ethyl acetate (50 mL), dried over Na₂SO₄,filtered, and concentrated. The residue was dissolved in MeOH (2 mL),Then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes. Then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 254 nm) to give example 117 as a white solid(32.8 mg).

Example 118

Compound (118-2) (72 mg, 0.3 mmol) and DBU (0.153 g, 1 mmol) in THF (5mL) was added into a solution of (113-5) (1 mL, 0.2 mmol) in toluene.The mixture was stirred at rt overnight. The mixture was quenched withwater, extracted with ethyl acetate (20 mL*3), dried over Na₂SO₄,filtered, and concentrated. The residue was dissolved in MeOH (2 mL),then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes, then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 254 nm) to give the example 118 as a white solid(40 mg).

Example 119

1. Synthesis of Compound (119-1)

A mixture of N,N-dimethylaniline (0.5 g, 4.13 mmol) andbistrimethylsilyl sulfate (0.5 g, 4.13 mmol) was heated at 160° C. for 5hours. The mixture was allowed to cool to rt and the resulting solid wasisolated by filtration and washed with Et₂O. The solid was thendissolved in H₂O, and the solution was concentrated in vacuo to give thetitle compound 600 mg (crude) as a white solid.

2). Synthesis of Compound (119-2)

Compound (119-1) (600 mg) was added portionwise to a suspension of PCl₅(931 mg, 4.5 mmol) in DCM (20 mL) at 0° C. The mixture was then allowedto warm to rt and was then stirred at rt for 3 h. The mixture wasconcentrated in vacuo and the residue was dissolved in Et₂O and H₂O. Thelayers were separated and the organic layer was dried over Na₂SO₄,filtered and concentrated in vacuo to give the title compound 320 mg asa yellow solid, which was used directly without further purification.

3). Synthesis of Compound (119-3)

Ammonia (10 mL) was added to a solution of (119-2) (320 mg) in THF (10mL) and was stirred at rt for 1.5 h, then concentrated. The residue waspurified by flash silica chromatography, elution gradient 40 to 100%EtOAc in petroleum ether to give the title compound (160 mg, 54.7%) as ayellow solid.

4). Synthesis of Example 119

Compound (119-3) (100 mg, 0.5 mmol) and DBU (76 mg, 0.5 mmol) in THF (5mL) was added into a solution of the compound (2a) (1 mL, 0.2 mmol) intoluene. The mixture was stirred at RT for overnight. The mixture wasquenched with water, extracted with ethyl acetate (50 mL), dried,filtered, and concentrated. The residue was dissolved in MeOH (2 mL),then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes, then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 220 nm) to give the example 119 (24.3 mg) as awhite solid.

Example 121

Compound (121-2) (72 mg, 0.3 mmol) and DBU (0.153 g, 1 mmol) in THF (1mL) was added into a solution of (93-5) (1 mL, 0.2 mmol) in toluene. Themixture was stirred at rt overnight. The mixture was quenched withwater, extracted with ethyl acetate (50 mL), dried over Na₂SO₄,filtered, and concentrated. The residue was dissolved in MeOH (2 mL).Then 1 drop of 37% HCl was added. The mixture was stirred at rt for 10minutes. Then was diluted with ethyl acetate (50 mL) and washedsequentially with saturated sodium bicarbonate and brine. The organiclayer was dried over Na₂SO₄, filtered and concentrated. The residue waspurified by Flash-Prep-HPLC ((IntelFlash-1): Column, C18; mobile phase,MeCN/H₂O, Detector, UV 254 nm) to give example 121 as a white solid(29.7 mg). The following desired examples were prepared using proceduressimilar to that described above. The MS data and 1H NMR data aredelineated in Table 11.

TABLE 11 Example MS data # Structure (M − 1)⁻ ¹H NMR (500 MHz) 12

643.31 13

587.29 (CDCl₃) 7.69 (2H, br s), 7.45 (2H, m), 7.10 (1H, br s), 6.52 (1H,s), 3.71 (1H, s), 3.41 (1H, br s), 3.30 (1H, br s), 3.17 (1H, br s),2.45 (3H, s), 0.95 (3H, d, J = 5.5 Hz), 0.90 (3H, s), 0.65 (3H, s). 14

641.24 (CDCl₃) 8.15 (1H, br s), 8.04 (2H, d, J = 8.0 Hz), 7.82 (2H, d, J= 8.0 Hz), 6.43 (1H, s), 3.71 (1H, s), 3.42 (1H, br s), 3.30 (1H, br s),3.18 (1H, br s), 0.95 (3H, d, J = 5.5 Hz), 0.90 (3H, s), 0.64 (3H, s).15

603.25 (CDCl₃) 7.82 (2H, d, J= 9.0 Hz), 7.41 (1H, br s), 6.99 (2H, d, J= 9.0 Hz), 6.48 (1H, s), 3.89 (3H, s), 3.71 (1H, s), 3.41 (1H, br s),3.29 (1H, br s), 3.17 (1H, br s), 0.95 (3H, d, J = 5.5 Hz), 0.90 (3H,s), 0.65 (3H, s). 16

605.26 (CDCl₃) 7.76 (1H, br s), 7.58 (1H, d, J = 9.5 Hz), 7.53 (1H, d, J= 9.5 Hz), 7.37 (1H, t, J = 7.5 Hz), 6.47 (1H, s), 3.71 (1H, s), 3.41(1H, br s), 3.30 (1H, br s), 3.18 (1H, br s), 2.37 (3H, s), 0.95 (3H, d,J = 5.5 Hz), 0.90 (3H, s), 0.65 (3H, s). 17

601.29 (CDCl₃) 7.63 (1H, s), 7.60 (1H, d, J = 7.0 Hz), 7.29 (1H, d, J =7.0 Hz), 7.18 (1H, br s), 6.51 (1H, s), 3.71 (1H, s), 3.41 (1H, br s),3.29 (1H, br s), 3.17 (1H, br s), 2.35 (3H, s), 2.33 (3H, s), 0.95 (3H,d, J = 5.5 Hz), 0.90 (3H, s), 0.65 (3H, s). 18

671.44 19

637.41 20

551.45 21

579.21 (CDCl₃) 7.66 (2H, m), 7.41 (1H, br s), 7.11 (1H, m), 6.48 (1H,s), 3.68 (1H, s), 3.39 (1H, br s), 3.31 (1H, br s), 3.19 (1H, br s),2.37 (3H, s), 0.94 (3H, d, J = 5.5 Hz), 0.90 (3H, s), 0.64 (3H, s). 22

579.30 (CDCl₃) 7.05 (1H, br s), 6.49 (1H, s), 3.68 (1H, s), 3.38 (1H, brs), 3.30 (1H, br s), 3.19 (1H, br s), 3.05 (1H, m), 2.18 (2H, m), 0.95(3H, d, J = 5.5 Hz), 0.90 (3H, s), 0.64 (3H, s). 23

617.25 (CDCl₃) 7.43 (1H, d, J = 9.5 Hz), 7.35 (1H, br s), 7.24 (1H, s),6.87 (1H, d, J = 9.0 Hz), 6.44 (1H, s), 6.09 (2H, s), 3.68 (1H, s), 3.38(1H, br s), 3.29 (1H, br s), 3.16 (1H, br s), 0.93 (3H, d, J = 5.5 Hz),0.88 (3H, s), 0.64 (3H, s). 24

607.23 (CDCl₃) 8.00 (1H, br s), 7.86 (1H, s), 7.76 (1H, d, J = 7.5 Hz),7.60 (1H, d, J = 8.0 Hz), 7.47 (1H, t, J = 8.0 Hz), 6.45 (1H, s), 3.69(1H, s), 3.39 (1H, br s), 3.29 (1H, br s), 3.17 (1H, br s), 0.93 (3H, d,J = 5.5 Hz), 0.88 (3H, s), 0.63 (3H, s). 25

607.22 (CDCl₃) 7.82 (1H, br s), 7.82 (2H, d, J = 7.5 Hz), 7.50 (2H, d, J= 7.5 Hz), 6.41 (1H, s), 3.69 (1H, s), 3.40 (1H, br s), 3.28 (1H, br s),3.16 (1H, br s), 0.93 (3H, d, J = 6.0 Hz), 0.88 (3H, s), 0.63 (3H, s).26

643.34 27

565.30 (CDCl₃) 7.11 (1H, br s), 6.52 (1H, s), 3.71 (1H, s), 3.66 (1H,m), 3.41 (1H, br s), 3.34 (1H, br s), 3.22 (1H, br s), 2.07 (4H, m),0.98 (3H, d, J = 5.5 Hz), 0.90 (3H, s), 0.66 (3H, s). 28

553.30 (CDCl₃) 7.06 (1H, br s), 6.69 (1H, s), 3.71 (1H, s), 3.41 (1H, brs), 3.33 (1H, br s), 3.21 (1H, br s), 1.47 (9H, s), 0.97 (3H, d, J = 6.5Hz), 0.90 (3H, s), 0.66 (3H, s). 29

543.25 (M − 1 + HCO₂H) 30

569.26 (M − 1 + HCO₂H) 31

559.26 32

609.27 33

661.32 (M − 1 + HCO₂H) 34

643.23 35

593.33 36

637.30 37

582.29 (CDCl₃) 7.31 (1H, br s), 6.33 (1H, s), 3.78 (4H, s), 3.71 (1H,s), 3.41 (1H, br s), 3.34 (1H, br s), 3.26 (4H, s), 3.21 (1H, br s),0.98 (3H, d, J = 6.5 Hz), 0.90 (3H, s), 0.66 (3H, s). 38

623.28 (CDCl₃) 8.54 (1H, d, J = 9.0 Hz), 8.22 (1H, d, J = 7.5 Hz), 8.14(1H, d, J = 8.0 Hz), 7.98 (1H, d, J = 7.0 Hz), 7.71 (1H, br s), 7.69(1H, t, J = 7.0 Hz), 7.64 (1H, t, J = 9.0 Hz), 7.55 (1H, t, J = 7.5 Hz),6.52 (1H, s), 3.71 (1H, s), 3.41 (1H, br s), 3.26 (1H, br s), 3.14 (1H,br s), 0.91 (3H, d, J = 6.0 Hz), 0.90 (3H, s), 0.62 (3H, s). 39

580.32 (CDCl₃) 7.30 (1H, br s), 6.40 (1H, s), 3.71 (1H, s), 3.41 (1H, brs), 3.34 (1H, br s), 3.26 (4H, s), 3.23 (5H, m), 1.68 (4H, br s), 0.98(3H, d, J = 6.5 Hz), 0.90 (3H, s), 0.66 (3H, s). 40

607.23 41

611.31 (M − 1 + HCO₂H) 42

612.30 (M − 1 + HCO₂H) 43

614.30 (M − 1 + HCO₂H) 44

636.44 45

593.33 46

551.29 47

594.31 48

566.29 49

574.36 50

657.35 51

591.26 52

596.44 53

629.43 54

649.37 55

613.40 56

633.29 (M − 1 + HCO₂H) 57

627.38 58

631.37 59

655.45 60

647.41 61

647.41 62

656.31 63

587.28 64

575.26 65

608.35 66

616.36 67

636.47 68

578.39 69

645.33 70

673.42 71

670.37 72

654.39 73

670.29 74

622.32 75

610.33 76

591.41 77

645.34 78

596.40 79

668.30 80

670.28 81

638.36 82

610.39 83

630.36 84

639.45 85

665.43 86

606.42 87

568.40 88

635.48 89

615.39 90

553.37 91

540.37 92

663.29 93

587.38 94

591.35 95

541.37 96

608.44 97

608.41 98

594.34 99

594.34 100

580.23 101

607.44 102

609.26 103

621.28 104

565.24 105

644.35 106

681.29 107

624.35 108

655.32 109

591.36 110

601.37 111

635.45 112

M + 1, 618.50 113

641.50 114

641.00 115

615.55 116

643.45 117

603.35 118

M + 1, 644.40 119

616.55 120

617.33 121

M + 1, 644.60 122

M + 1, 571.45 123

631.34 124

512.31 125

526.33 126

622.31 127

645.46 128

556.34

ASSAYS

Human FXR (NR1H4) Assay

Determination of a ligand mediated Gal4 promoter driven transactivationto quantify ligand binding mediated activation of FXR, FXR ReporterAssay kit purchased from Indigo Bioscience (Catalogue number: IB00601)to determine the potency and efficacy of compound developed by Enantathat can induce FXR activation. The principle application of thisreporter assay system is to quantify functional activity of human FXR.The assay utilizes non-human mammalian cells, CHO (Chinese hamsterovary) cells engineered to express human NR1H4 protein (referred to asFXR). Reporter cells also incorporate the cDNA encoding beetleluciferase which catalyzes the substrates and yields photon emission.Luminescence intensity of the reaction is quantified using aplate-reading luminometer, Envision. Reporter Cells include theluciferase reporter gene functionally linked to an FXR responsivepromoter. Thus, quantifying changes in luciferase expression in thetreated reporter cells provides a sensitive surrogate measure of thechanges in FXR activity. EC₅₀ and efficacy (normalize to CDCA set as100%) is determined by XLFit. The assay is according to themanufacturer's instructions. In brief, the assay was performed in white,96 well plates using final volume of 100 ul containing cells withdifferent doses of compounds. Retrieve Reporter Cells from −80° C.storage. Perform a rapid thaw of the frozen cells by transferring a 10ml volume of 37° C. cell recovery medium into the tube of frozen cells.Recap the tube of Reporter Cells and immediately place it in a 37° C.water bath for 5-10 minutes. Retrieve the tube of Reporter CellSuspension from the water bath. Sanitize the outside surface of the tubewith a 70% alcohol swab, and then transfer it into the cell culturehood. Dispense 90 μl of cell suspension into each well of the 96-wellAssay Plate. Transfer the plate into 37° C. incubator, allowing thecells adherent to the bottom of the well. Dilute compounds in DilutionPlate (DP), and administrate to cells at Assay Plate (AP). DMSO contentof the samples was kept at 0.2%. Cells were incubated for additional 22hours before luciferase activities were measured. Thirty minutes beforeintending to quantify FXR activity, remove Detection Substrate andDetection Buffer from the refrigerator and place them in a low-lightarea so that they may equilibrate to room temperature. Remove theplate's lid and discard all media contents by ejecting it into anappropriate waste container. Gently tap the inverted plate onto a cleanabsorbent paper towel to remove residual droplets. Cells will remaintightly adhered to well bottoms. Add 100 μl of luciferase detectionreagent to each well of the assay plate. Allow the assay plate to restat room temperature for at least 5 minutes following the addition ofLDR. Set the instrument (Envision) to perform a single 5 second “plateshake” prior to reading the first assay well. Read time may be 0.5second (500 mSec) per well. EC₅₀ and Efficacy (normalize to CDCA set as100%) is determined by XLFit.

In Vitro Human TGR5 (GPBAR1) Activity Assay

The potency and efficacy of the compounds of the invention on TGR5receptor was evaluated using in vitro assays which carried out using theexpress kit from DiscoverX (cAMP HUNTER™ eXpress GPBAR1 CHO-K1 GPCRAssay; Cataloguer number: 95-0049E2CP2S)GPBAR1 (G protein-coupled bileacid receptor 1) encodes a member of the G protein-coupled receptor(GPCR) superfamily. GPBAR1 activation following ligand binding initiatesa series of second messenger cascades that result in a cellularresponse. Treatment of CHO cells expressing GPBAR1 with bile acidsinduces the production of intracellular cAMP and internalization of thereceptor. The potency and efficacy of compound for GPBAR1 activation bymeasuring cyclic adenosine monophosphate (cyclic AMP or cAMP) levels inlive cells using a competitive immunoassay based on Enzyme FragmentComplementation (EFC).

In briefly, following seeding the cells into the white, 96 wellmicroplate, place it in a 37° C., 5% CO₂ in a humidified incubator for18-24 hours prior to testing. On second day, proceed to the appropriatecAMP HUNTER™ eXpress Protocol according to the manufacturer'sinstructions. Dissolve agonist compound in DMSO at the desired stockconcentration, and prepare 3-fold serial dilutions of agonist compoundin Cell Assay Buffer. The concentration of each dilution should beprepared at 4× of the final screening concentration (i.e. 15 μLcompound+45 μL Cell Assay Buffer/cAMP Antibody Reagent). For eachdilution, the final concentration of solvent should remain constant.Transfer 15 μL diluted compound the assay plate and incubate the platefor 30 minutes at 37° C. Following agonist incubation, add 60 μL ofworking cAMP detection reagents/cAMP Solution mixture (cAMP LysisBuffer, Substrate Reagent 1, cAMP Solution D) to the appropriate wells.Incubate for 1 hour at room temperature (23° C.), protected from light.Add 60 μl of cAMP Solution A to the appropriate wells. Incubate for 3hours at room temperature (23° C.), protected from light. Read sampleson Envision standard luminescence plate reader. Calculate of averageEC₅₀ after logarithm transformation.

To assess the FXR agonistic potency of the example compounds as well asfor reference compound, potency ranges were determined in the Human FXR(NR1H4) Assay as listed below in Table 12. The efficacy was normalizedto CDCA set as 100%. (A=EC50<0.1 μM; B=0.1 μM<EC50<1.0 μM; C=1.0μM<EC50<10 μM; D=EC50>10 μM)

TABLE 12 Example EC50 (uM) Efficacy (%) CDCA D 100 6-ECDCA B 223 1 A 3372 A 300 3 A 396 4 A 342 5 B 369 6 B 269 7 C 259 8 n/a n/a 10 B 168 11 C267 12 A 327 13 A 245 14 B 295 15 A 337 16 A 265 17 A 357 18 A 319 19 B323 20 B 334 21 B 402 22 A 477 23 A 488 24 A 421 25 A 357 26 B 378 27 A275 28 A 323 29 C 299 30 C 194 31 B 305 32 A 309 33 A 270 34 A 219 35 B217 36 B 228 37 A 254 38 A 231 39 A 379 40 B 244 41 B 350 42 B 343 43 C264 44 na 1 45 C 178 46 A 315 47 A 361 48 A 420 49 C 236 50 A 309 51 A319 52 B 281 53 A 263 54 A 273 55 A 271 56 B 295 57 A 297 58 A 307 59 B344 60 A 346 61 A 403 62 C 476 63 B 452 64 C 334 65 A 498 66 A 375 67 A351 68 A 458 69 A 490 70 A 434 71 B 419 72 B 384 73 B 444 74 B 242 75 A323 76 A 290 77 C 89 78 C 210 79 B 249 80 B 295 81 A 315 82 A 321 83 C348 84 B 351 85 B 324 86 A 390 87 B 343 88 A 378 89 A 351 90 A 407 91 A399 92 C 325 93 A 443 94 A 361 95 C 245 96 A 387 97 A 308 98 A 344 99 A266 100 C 364 101 A 244 102 A 210 103 A 328 104 C 298 105 B 322 106 A281 107 B 311 108 B 203 109 B 219 110 C 320 111 A 314 112 A 287 113 A366 114 A 322 115 B 291 116 A 322 117 A 305 118 A 267 119 A 363 120 C198 121 B 199 122 C 121 123 B 271 124 C 195 125 C 256 126 C 238 127 C210 128 C 405 129 n/a n/a 130 C 146 131 C 8 132 C 7 133 D 80 134 n/a 19135 C 4 136 C 159 137 C 105 138 C 2 139 C 138 140 n/a 1 141 C 16 142 n/a7 143 n/a 6

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A compound represented by Formula (VI-A) or(VI-B), or a pharmaceutically acceptable salt thereof:

wherein: m is 0, 1, 2 or 3; and R₁₀ is selected from hydrogen,substituted or unsubstituted —C₁-C₈ alkyl, substituted or unsubstituted—C₂-C₈ alkenyl, substituted or unsubstituted —C₂-C₈ alkynyl, andsubstituted or unsubstituted —C₃-C₈ cycloalkyl.
 2. The compound of claim1, wherein m is 1 and R₁₀ is selected from the group consisting ofhydrogen, methyl, ethyl, isopropyl, butyl, t-butyl, propyl, benzyl,vinyl, allyl, CF₃,


3. A pharmaceutical composition comprising a compound according to claim1 and a pharmaceutically acceptable carrier.
 4. A method forameliorating a disease or condition selected from the group consistingof primary biliary cirrhosis, cerebrotendinous xanthomatosis, primarysclerosing cholangitis, alcoholic liver disease, nonalcoholic fattyliver disease, nonalcoholic steatohepatitis, atherosclerosis,hypercholesterolemia, hypertriglyceridemia, Type II diabetes, andhepatocellular carcinoma in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of acompound according to claim
 1. 5. The method according to claim 4,wherein the disease or condition is selected from the group consistingof primary biliary cirrhosis (PBC), nonalcoholic fatty liver disease(NAFLD), and nonalcoholic steatohepatitis (NASH).